Purpose: It is still unknown which metabolite of isopropyl unoprostone is responsible for reducing intraocular pressure. This study was carried out to measure intraocular metabolites of isopropyl unoprostone in the aqueous humor of primate and human eyes. Methods: Nine monkeys were randomly divided into three groups, all of which received isopropyl unoprostone. In group I, the drug was scheduled to be instilled at 0 hr, in group II at 1 hr, and in group III at 2 hr, prior to aqueous humor aspiration in order to determine metabolite concentration. Furthermore, 27 patients scheduled for cataract surgery and intraocular lens implantation were divided into five groups that received isopropyl unoprostone. In group A, the drug was scheduled to be instilled at 0 hr, in group B at 1 hr, in group C at 2 hr, in group D at 3 hr, and in group E at 4 hr, prior to surgery. At the beginning of the operation, the aqueous humor was aspirated. Metabolites of isopropyl unoprostone in the aqueous humor were determined by high-performance liquid chromatography. Results: M1 (3-[(1R,2R,3R,5S)-3,5-dihydroxy-2-(3-oxodecyl)cyclopentyl]propionic acid) (unoprostone free acid) and M2 ((Z)-7-[(1R,2R,3R,5S)-3,5-dihydroxy-2-(3-oxodecyl)cyclopentyl]hept-5-enoic acid), an intraocular oxidized metabolite of isopropyl unoprostone, were measured. M1:M2 in monkeys was respectively 0:0 ng/ml in group I, 150.2 ± 45.1:9.5 ± 1.7 (p < 0.05) in group II, and 74.6 ± 31.4:19.2 ± 5.3 (p < 0.01) in group III. M1:M2 in humans was respectively 0:0 ng/ml in group A, 50.6 ± 22.3:3.2 ± 1.3 (p < 0.05) in group B, 125.0 ± 23.1:12.2 ± 3.4 (p < 0.001) in group C, 144.9 ± 33.8:24.5 ± 6.2 (p < 0.01) in group D, and 56.7 ± 21.5:18.7 ± 5.3 (p < 0.05) in group E. Conclusions: A free acid of isopropyl unoprostone is the major intraocular metabolite of isopropyl unoprostone that is expected to act on target tissues in the eyes of both monkeys and humans.