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Short answer: oligomycin is an inhibitor of mitochondrial ATP synthase. Oligomycin A has the formula C45H74O11 and a molecular weight of 791.1 g/mol (PubChem). In respirometry it is used to separate oxygen consumption that drives ATP production from oxygen consumption that leaks protons. This page is general reference for researchers and lab staff, not safety or medical advice.
What is oligomycin?
Oligomycin is a small-molecule inhibitor of ATP synthase, the mitochondrial complex that makes ATP from the proton gradient across the inner membrane. Labs mostly meet it as a reagent injected during Seahorse XF Cell Mito Stress Tests, where it is the first compound added after the basal oxygen consumption rate (OCR) reading. Searches for “oligomycin” usually come from people who need one of three things: the mechanism, the molecular weight, or the role it plays in an assay.
Oligomycin mechanism of action
ATP synthase lets protons flow back into the mitochondrial matrix and uses that flow to make ATP. Oligomycin blocks this. The 2018 cryo-EM study by Srivastava and colleagues (Science 360, DOI 10.1126/science.aas9699; deposited as RCSB PDB entry 6CP5, 4.20 Å resolution) determined structures of the yeast (Saccharomyces cerevisiae) ATP synthase Fo region with oligomycin bound, and reports that they show how oligomycin blocks ATP synthesis.
The Gu et al. STAR Protocols paper (PMC7797920) describes the downstream effect in cells: by inhibiting ATP synthase, oligomycin can rapidly hyperpolarize the mitochondrial membrane and prevent protons from passing through the complex. Because that proton flow is shut off, the oxygen consumption that was coupled to ATP production falls.
Oligomycin molecular weight and formula
PubChem lists oligomycin A (CID 52947716) with molecular formula C45H74O11 and molecular weight 791.1 g/mol. That figure belongs to oligomycin A specifically. When you calculate a molar concentration, check which compound or mixture your supplier actually supplied and use the molecular weight on the certificate of analysis or product record for that lot, rather than assuming 791.1.
How oligomycin is used in a Mito Stress Test
In Agilent’s XF Cell Mito Stress Test guidance, oligomycin is injected first after basal respiration. The drop in OCR after injection is read as ATP-linked respiration, and the OCR that remains is read as proton leak, meaning respiration not coupled to ATP production. Later injections resolve the other parameters; the Seahorse assay guide walks through the full injection sequence and what each parameter means, and the rotenone guide covers the complex I inhibitor used later in the run.
Checklist: what to confirm before trusting oligomycin-derived numbers
The STAR Protocols paper raises several points that affect how oligomycin results should be read. They are collected here as a pre-analysis checklist.
- Dose. The protocol gives 2 µM oligomycin as an example that it says can meet most cell types, and advises titrating when the OCR change after injection is minimal. Treat any single concentration as a starting point for your cell type, not a constant.
- Access to the target. In intact cells the compound has to reach the mitochondria, so drug access and permeability can limit the apparent effect.
- Assay medium. Substrates in the medium (the paper names pyruvate, glutamine and glucose) can affect the measurement, so compare conditions only when the medium is the same.
- Molecular weight and lot. Confirm the compound identity and molecular weight for your lot before preparing stock concentrations.
Handling and hazard
The GHS classification recorded in PubChem for oligomycin A is signal word Warning, hazard statement H302 (harmful if swallowed), with the irritant (GHS07) pictogram. Follow your institution’s chemical hygiene plan and the supplier’s current safety data sheet for storage, solvent and disposal details.
Common misunderstandings
- “A drop in oxygen use after oligomycin means the cells are dying.” In a Mito Stress Test the drop is the expected result of blocking ATP synthase; its size is read as ATP-linked respiration.
- “The remaining OCR is zero respiration.” The remainder after oligomycin is read as proton leak; the later injections separate other components.
- “791.1 applies to every oligomycin product.” It is the PubChem value for oligomycin A; check the lot documentation.
- “One concentration works everywhere.” The 2 µM figure is an example, with titration advised when the response is minimal.
FAQ
What is oligomycin?
Oligomycin is an inhibitor of mitochondrial ATP synthase used as a research reagent, most often in Seahorse XF Mito Stress Tests to estimate ATP-linked respiration and proton leak.
What is the mechanism of action of oligomycin?
It blocks ATP synthesis by ATP synthase; cryo-EM structures of the yeast Fo region with oligomycin bound (PDB 6CP5) show how, and in cells the block hyperpolarizes the mitochondrial membrane.
What is the molecular weight of oligomycin?
Oligomycin A is 791.1 g/mol with formula C45H74O11 (PubChem CID 52947716). Check your supplier’s lot documentation for the product you hold.
Sources
- PubChem, oligomycin A, CID 52947716 (properties and GHS classification), pubchem.ncbi.nlm.nih.gov, read 2026-11-08.
- RCSB Protein Data Bank entry 6CP5, rcsb.org/structure/6CP5, read 2026-11-08; Srivastava AP et al., Science 360, 2018, DOI 10.1126/science.aas9699.
- Gu X, Ma Y, Liu Y, Wan Q. STAR Protocols, PMC7797920 (Seahorse XF Cell Mito Stress Test protocol), ncbi.nlm.nih.gov/pmc/articles/PMC7797920, read 2026-11-08.
- Agilent Seahorse XF Cell Mito Stress Test documentation, agilent.com (injection order and parameter definitions).
