This study was performed to clarify the ocular hypotensive mechanism of topical isopropyl unoprostone (unoprostone), a novel prostaglandin (PG) metabolite-related drug, in the rabbit eye. The intraperitoneal administration of indomethacin (50 mg/kg) 1 hour before administration of topical 0.12% unoprostone partially diminished the intraocular pressure (IOP) reduction, and completely blocked the increase in aqueous PGE2 concentration caused by unoprostone. Aqueous humor dynamics measurements in the unoprostone- and the vehicle-treated contralateral eyes with indomethacin pretreatment revealed that aqueous flow determined by fluorophotometry was not significantly different, 2.3 +/- 0.3 and 2.4 +/- 0.2 microliters/min, respectively; the total outflow facility measurements determined by the two-level constant pressure perfusion method were 0.20 +/- 0.01 and 0.14 +/- 0.01 microliters/min/mmHg, respectively (p < 0.05); the uveoscleral outflow measurements determined by the fluorescein isothiocyanate-dextran perfusion method were 0.49 +/- 0.02 and 0.46 +/- 0.02 microliters/min, respectively (p < 0.05). The magnitude of the IOP reduction induced by unoprostone was estimated to be 5.2 mmHg, which agrees well with the actual IOP reduction. In conclusion, unoprostone lowers the IOP by affecting aqueous outflow pathways, primarily the pressure-dependent conventional pathway and the secondary uveoscleral outflow pathway in rabbits. Endogenous PGs induced by topical unoprostone are also involved in lowering the IOP in rabbits.