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Longevity

Methylene Blue

Also known as PROVAYBLUE · Methylthioninium chloride · Basic Blue 9

FDA-approvedAHuman trial evidenceWhat this means

Methylene blue is a thiazine dye and oxidation-reduction agent, not a peptide. It is FDA-approved as PROVAYBLUE injection for treating acquired methemoglobinemia in children and adults, with a boxed warning for serotonin syndrome when combined with serotonergic drugs or opioids, and it is contraindicated in G6PD deficiency. It has also been studied in small human trials, for example in septic shock and brain imaging; this entry summarizes the label and selected studies for research reference only.

US status
FDA-approved
Evidence
Grade A
Half-life
Approximately 24 hours in humans; about 40% excreted unchanged in urine 1
Class
Small molecule (not a peptide); thiazine (phenothiazinium) dye and oxidation-reduction agent
Studied in: Cognition & neuroprotection
More
Log a sample
Dosing referenceCycle-length evidence
Unbranded reference vial illustrating Methylene Blue; not an actual product
Research reference, not advice. Label quotes, not a recommendation. Amiravo sells nothing; some links are referrals.Read the full notice

Key takeaways

  • Methylene blue is an FDA-approved oxidation-reduction agent (PROVAYBLUE) for acquired methemoglobinemia in children and adults; it is not a peptide 1.
  • The label carries a boxed warning: serious or fatal serotonin syndrome can occur with serotonergic drugs and opioids, and concomitant use with SSRIs, SNRIs, MAOIs and opioids should be avoided 1.
  • It is contraindicated in G6PD deficiency because of the risk of hemolytic anemia, and in severe hypersensitivity to thiazine dyes 1.
  • In the label's clinical studies, 26 of 28 adults with a baseline measurement had at least a 50% reduction in methemoglobin at the first post-dose assessment 1.
  • In a randomized trial in 91 patients with septic shock, early adjunctive methylene blue shortened time to vasopressor discontinuation (69 vs 94 hours) 4.

Development stage

Where Methylene Blue sits between laboratory work and regulatory approval.

  1. Preclinical
  2. Phase 1
  3. Phase 2
  4. Phase 3
  5. Approved (current)

Stage comes from the regulatory record and ClinicalTrials.gov studies naming the compound (for compounds approved nowhere, only completed trials or trials with posted results count) (checked 2026-10-24). A registered trial is not a result.

Mechanism of action

Methylene blue promotes non-enzymatic redox conversion of methemoglobin to hemoglobin: it is first converted to leucomethylene blue by NADPH reductase, and leucomethylene blue reduces the ferric iron of methemoglobin to the ferrous state 1.

Monoamine oxidase inhibition

The label cites literature describing methylene blue as a potent reversible inhibitor of monoamine oxidase, which underlies the risk of serotonin syndrome with serotonergic drugs 1.

G6PD deficiency

Patients with G6PD deficiency may not reduce methylene blue to its active form and are at risk of severe hemolysis 1.

Molecular structure

PubChem CID 6099 ↗
2D chemical structure of Methylene Blue

2D depiction generated from the PubChem record. Depictions are schematic and do not show salt form, counter-ions or conformation.

Formula
C16H18ClN3S
Molecular weight
319.9 g/mol
CAS
61-73-4
InChIKey
CXKWCBBOMKCUKX-UHFFFAOYSA-M

Research overview

Human research

The approval was based on 31 adults with acquired methemoglobinemia in an open-label single-arm study and an observational registry, plus pediatric case series 1. In a single-centre randomized trial of 91 patients with septic shock, methylene blue started within 24 hours shortened time to vasopressor discontinuation and ICU and hospital stay, with similar mortality 4. In 26 healthy adults, a single low oral dose increased fMRI responses during attention and short-term memory tasks and was associated with a 7% increase in correct memory-retrieval responses 3.

Animal research

In two-year studies, oral methylene blue caused pancreatic islet adenomas or carcinomas in male rats but no drug-related neoplasms in mice; it produced adverse developmental outcomes in rats and rabbits at oral exposures well above the clinical dose 1.

In-vitro research

Methylene blue was genotoxic in the Ames test and in in vitro sister chromatid exchange and chromosomal aberration tests, but negative for micronucleus induction in mice; in vitro it reduced human sperm motility and inhibited several CYP and UGT enzymes 1.

Key studies

  1. PROVAYBLUE label, Clinical Studies (NCT03395223, NCT03542760) (2025)
    Prospective open-label single-arm study and prospective multicenter observational registry
    Model: Humans (adults with acquired methemoglobinemia, n=31)

    Of 28 subjects with a baseline methemoglobin value (mean 18.4%), 26 (92.9%) had at least a 50% reduction at the first post-dose assessment (median 2.7 hours) 1.

  2. Ibarra-Estrada et al., Crit Care (2023)
    Single-centre randomized placebo-controlled trial, 91 patients (NCT04446871)
    Model: Humans (patients with septic shock, Sepsis-3)

    Time to vasopressor discontinuation was 69 versus 94 hours; vasopressor-free days, ICU stay and hospital stay favoured methylene blue; mortality and ventilator days were similar; no serious adverse effects related to methylene blue were reported 4.

  3. Rodriguez et al., Radiology (2016)
    Randomized, double-blind, placebo-controlled fMRI study before and 1 hour after a single low oral dose
    Model: Humans (healthy adults aged 22 to 62, n=26)

    Methylene blue increased insular cortex response during a sustained-attention task and prefrontal, parietal and occipital responses during a short-term memory task, with a 7% increase in correct retrieval responses 3.

Dosing as stated on the FDA label

Quoted from the prescribing information for reference. Prescribing decisions belong to a licensed clinician; this is not a recommendation or instruction for use.

Full dosing reference, study durations and vial arithmetic →
  • PROVAYBLUE for acquired methemoglobinemia: administer 1 mg/kg intravenously over 5 to 30 minutes. If the methemoglobin level remains greater than 30% or if clinical signs and symptoms persist, a repeat dose of 1 mg/kg may be given one hour after the first dose. If methemoglobinemia does not resolve after 2 doses, consider initiating alternative interventions. 1
  • Renal impairment: the recommended dosage in patients with moderate or severe renal impairment (eGFR 15 to 59 mL/min/1.73 m2) is a single dose of 1 mg/kg; no adjustment is recommended for mild renal impairment. 1
  • Per the label, the recommended dose is 1 mg/kg (lower or greater doses are not recommended), the maximum recommended number of doses is two, ensure patent venous access, do not administer subcutaneously, and the injection may be diluted in 50 mL 5% Dextrose Injection (avoid sodium chloride solutions). 1
  • Dosage forms per the label: 50 mg/10 mL and 10 mg/2 mL (5 mg/mL) single-dose ampules or vials; strength is expressed in terms of the trihydrate. 1
  • In the label's clinical studies, most doses administered were 1 mg/kg (82.9%), with doses from 0.78 mg/kg to 2 mg/kg; two patients received two doses and one received three 1.
  • In SHOCKEM-Blue (NCT04446871), patients with septic shock received an intravenous infusion of 100 mg methylene blue in 500 mL of 0.9% sodium chloride over 6 hours once a day for 3 doses, or a saline placebo infusion 4,5.
  • In the fMRI study, healthy adults received a single low oral dose of methylene blue or placebo, with imaging 1 hour later; the amount is not stated in the abstract 3.

Published adverse-event data

Reported in the cited trials or product labels. Frequencies apply to the stated population and do not transfer to other settings.

Label warnings

  • Boxed warning: PROVAYBLUE may cause serious or fatal serotonergic syndrome when used in combination with serotonergic drugs and opioids; avoid concomitant use with SSRIs, SNRIs, MAOIs and opioids. The label advises patients not to take serotonergic drugs within 72 hours after the last dose. 1
  • Contraindications: severe hypersensitivity reactions to methylene blue or any other thiazine dye; glucose-6-phosphate dehydrogenase (G6PD) deficiency, due to the risk of hemolytic anemia. 1
  • Lack of effectiveness: methemoglobinemia may not resolve or may rebound in patients with methemoglobinemia due to aryl amines (such as aniline) or sulfa drugs (such as dapsone), and patients with G6PD deficiency may not reduce PROVAYBLUE to its active form. 1
  • Interference with monitoring: methylene blue may cause underestimation of oxygen saturation by pulse oximetry and a fall in Bispectral Index readings; it may also interfere with urine tests that rely on a blue indicator. 1
  • Pregnancy and lactation: PROVAYBLUE may cause fetal harm; breast-feeding should be discontinued during and for up to 8 days after treatment. 1
  • Confusion, dizziness and visual disturbances may occur; patients should not drive or operate machinery until these have resolved. 1
  • Headache, hypokalemia, diarrhea, hypomagnesemia, myoclonus, nausea, seizure-like phenomena
    2% or more (each)
    31 adults with acquired methemoglobinemia (label) 1 · Serious adverse reactions in 3.2%, including one seizure-like phenomenon
  • Serotonin syndrome
    Reported with methylene blue class products, mostly with serotonergic drugs; some fatal
    Label boxed warning 1 · Serious or fatal
  • Hemolytic anemia
    Can occur; onset may be delayed 1 or more days
    Label warnings; severe in G6PD deficiency 1 · May require transfusion
  • Anaphylaxis and severe hypersensitivity
    Reported with methylene blue class products
    Label warnings 1 · Serious
  • Hypotension, wheezing, reduced oxygenation
    Reported at single doses of 3 mg/kg or more of methylene blue class products
    Label overdosage section 1 · Serious
  • Blue discoloration of skin and body fluids; phototoxicity
    Not quantified
    Label patient counseling and class adverse reactions 1 · Not graded

Emerging safety signals

  • Serotonin syndrome, including fatal cases, has been reported mostly with concomitant serotonergic drugs; the boxed warning was revised in 11/2023 per the label highlights (regulatory label evidence) 1. 1 (2023)
  • Methylene blue was genotoxic in several in vitro assays and caused pancreatic islet tumours in male rats in a two-year oral study (nonclinical label evidence) 1. 1 (2025)
  • Exposure rises substantially with renal impairment (AUC up by 52%, 116% and 192% in mild, moderate and severe impairment), and the label directs a single dose in moderate or severe impairment (label pharmacokinetic evidence) 1. 1 (2025)

What is not known: The exposure-response and exposure-safety relationships are unknown per the label 1. The approval rests on 31 adults in uncontrolled studies and small pediatric case series 1. Uses outside methemoglobinemia, including septic shock and cognition, are supported only by small single-centre or short-term trials 3,4. Fertility studies have not been conducted 1.

Clinicians and researchers on video

YouTube videos about Methylene Blue from clinicians and researchers. Their views, in their own words; not reviewed or endorsed by Amiravo. Compare what they say with the evidence above.

  • 👉 Methylene blue isn’t the miracle biohack people claim. Here’s the science behind it. 💊 #pharmacy
    Dr. Ethan Melillo, PharmD · 1.4M views

These creators are not affiliated with Amiravo and do not endorse Amiravo or any product linked on this site. Nothing here is medical advice. Video views counted on YouTube as of Oct 24, 2026.

Regulatory status

Status
Approved by the FDA as PROVAYBLUE (methylene blue) injection, NDA 204630, original approval 8 April 2016, for the treatment of pediatric and adult patients with acquired methemoglobinemia 1,2.
United States (FDA)
Approved under NDA 204630 (PROVAYBLUE, original approval 8 April 2016) per the Drugs@FDA application record 2; the current label was reviewed from DailyMed 1.
Sport (WADA)
Methylene blue is not named on the WADA 2026 Prohibited List 7.

Status varies by country and changes over time. How we describe regulatory status.

Quality and testing notes

Methylene blue is a small molecule. Pharmaceutical-grade (USP) injection should be distinguished from industrial, laboratory-stain or aquarium grades, which DailyMed shows are also marketed under 'methylene blue' names [1]. A certificate of analysis should state the hydrate form (the label expresses strength as the trihydrate; anhydrous molecular weight 319.86), assay, related substances including azure B (which the label identifies as a minor impurity and metabolite), heavy-metal content and, for injectables, sterility and endotoxin [1].

How purity testing works · Reading a certificate of analysis

Check a Methylene Blue certificate of analysis against its expected mass (319.9 Da)

Storage and handling

Per the PROVAYBLUE label: store at 20 to 25 degrees C (excursions 15 to 30 degrees C permitted); do not refrigerate or freeze; keep the ampule or vial in the original package to protect from light [1].

Frequently asked questions

Is methylene blue FDA-approved?
Yes. PROVAYBLUE injection (NDA 204630, 2016) is approved for acquired methemoglobinemia in pediatric and adult patients 1,2.
Is methylene blue a peptide?
No. It is a phenothiazinium dye with formula C16H18ClN3S 6.
Why does it have a boxed warning?
Because serious or fatal serotonin syndrome can occur with serotonergic drugs and opioids; it is described as a potent reversible MAO inhibitor 1.
Who should not receive it?
People with G6PD deficiency or severe hypersensitivity to thiazine dyes, per the label 1.
What is its half-life?
About 24 hours 1.
Has it been studied for brain function?
One randomized fMRI study in 26 healthy adults reported increased task-related brain activity and a 7% increase in correct memory-retrieval responses after a single low oral dose 3.
Does it affect pulse oximetry?
Yes, it may cause underestimation of oxygen saturation readings 1.

Latest indexed research

No new studies were filed in the last sweep.

Search all studies on Methylene Blue (PubMed, Europe PMC, ClinicalTrials.gov)

Listed by publication type from the index record. Inclusion is not an endorsement; open the source to read the findings.

Revision history

Meaningful changes to this entry, with dates and sources. Last reviewed Oct 23, 2026. Typo and layout fixes are not listed.

No recorded changes since the change log began on Oct 23, 2026.

Last reviewed October 23, 2026 by the Amiravo editorial team.

Read our editorial policy · Report an error on this page

Need the math?

Laboratory arithmetic with every formula shown.

For research use only. Not for human or veterinary use. Not a drug, food, or dietary supplement. This page is educational and is not medical advice.

Unbranded reference vial illustrating Methylene Blue; not an actual product

Unbranded reference image. Amiravo does not produce, supply or sell this compound.

Specifications

Molecular formula
C16H18ClN3S
Molecular weight
319.9 g/mol
CAS number
61-73-4
PubChem CID
6099
FDA UNII
8NAP7826UB
ChEMBL
CHEMBL405110
DrugBank
DB09241
Format
Bottle
Other forms seen
Oral solution (drops) 0.5% · Oral solution (drops) 1% (10 mg/mL) · Oral solution (drops) 2%
Type
Single compound

Identifiers from PubChem. Verify against the supplier’s documentation for your lot.

Research stats

How much has been published, not how good it is.

PubMed records
27,908
Indexed as human research
7,772
Animal-only research
4,842
Randomized controlled trials
398
Registered trials
343
Trials with posted results
32
FDA adverse-event reports (FAERS)
1,605
NIH-funded projects naming it
342
2006: 3722007: 4732008: 4772009: 5372010: 5542011: 5932012: 6532013: 7502014: 8222015: 9092016: 9392017: 10342018: 13502019: 14682020: 16282021: 17232022: 19532023: 20222024: 19832025: 19232026: 159920062026
PubMed records per year (current year is partial).

Counts from title/abstract searches of PubMed, ClinicalTrials.gov, openFDA and NIH RePORTER, checked 2026-10-24. Names shared with other substances can inflate counts. FAERS reports are spontaneous and do not establish cause. Funding shows interest, not evidence. Method

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