Purpose: To determine whether bimatoprost is hydrolyzed to its free acid after topical application in humans in vivo.Design: Prospective, masked, and vehicle controlled.Participants: Thirty-one eyes of 31 patients with cataracts.Methods: Beginning 7 days before scheduled cataract surgery, one eye of each patient was treated with bimatoprost 0.03% or vehicle once daily, with the last drop administered 2 to 12 hours before anterior chamber paracentesis before cataract surgery. In a masked fashion, aqueous humor specimens were assayed for bimatoprost and its free acid by high-pressure liquid chromatography and mass spectrometry.Main Outcome Measure: Detection of the free acid of bimatoprost in aqueous humor.Results: Aqueous humor concentrations of the free acid of bimatoprost were 22.0+/-7.0 nmol/l (mean+/- standard error of the mean, n = 12) and 7.0+/-4.6 nmol/l (n = 8) at 2 and 12 hours, respectively, and below the limit of detection after vehicle (n = 10). Concentrations of bimatoprost (amide) were 5.7 11.4 and 1.1+/-0.4 nmol/l at 2 and 12 hours, respectively, and undetectable after vehicle.Conclusion: After topical application of bimatoprost in humans, a sufficient concentration of its free acid, a potent FP prostanoid receptor agonist, is found in the aqueous humor to account for its ability to reduce intraocular pressure. Ophthalmology 2004;111:2193-2198 (C) 2004 by the American Academy of Ophthalmology.