AIM: To determine the aqueous humour concentration of the acid hydrolysis products of bimatoprost and latanoprost after a single topical dose of bimatoprost 0.03% or latanoprost 0.005% in humans. METHODS: Randomised, controlled, double-masked, prospective study. 48 eyes of 48 patients scheduled for routine cataract surgery were randomised in an 8:2:2 ratio to treatment with a single 30 mul drop of bimatoprost 0.03%, latanoprost 0.005% or placebo at 1, 3, 6 or 12 h before the scheduled cataract surgery. Aqueous humour samples were withdrawn at the beginning of the surgical procedure and analysed using high-performance liquid chromatography-tandem mass spectrometry. RESULTS: Bimatoprost acid (17-phenyl trinor prostaglandin F2alpha) was detected in aqueous samples at a mean concentration of 5.0 nM at hour 1, 6.7 nM at hour 3 and 1.9 nM at hour 6 after bimatoprost treatment. After latanoprost treatment, the mean concentration of latanoprost acid (13,14-dihydro-17-phenyl trinor prostaglandin F2alpha) in aqueous samples was 29.1 nM at hour 1, 41.3 nM at hour 3 and 2.5 nM at hour 6. Acid metabolites were below the limit of quantitation in all samples taken 12 h after dosing and in all samples from placebo-treated patients. None of the samples from latanoprost-treated patients contained quantifiable levels of non-metabolised latanoprost. Non-metabolised bimatoprost was detected in aqueous samples at a mean concentration of 6.6 nM at hour 1 and 2.4 nM at hour 3 after bimatoprost treatment. CONCLUSIONS: Low levels of bimatoprost acid were detected in aqueous humour samples from patients with cataract treated with a single dose of bimatoprost. Latanoprost acid concentrations in samples from patients treated with latanoprost were at least sixfold higher. These results suggest that bimatoprost acid in the aqueous humour does not sufficiently account for the ocular hypotensive efficacy of bimatoprost.