Intravitreal injection of purified human chorionic gonadotropin (hCG) in rabbits decreased intraocular pressure (IOP). A dose-dependent decrease in IOP was observed with intravitreal hCG concentrations at 30 nM and 100 nM. The onset of this effect was later than 10 hr following the injection and it lasted for more than 24 hrs. The purified beta-subunit of hCG caused a similar decrease in IOP with a short duration. The threshold intravitreal concentration was 10 nM. Unlike the intact hCG, the hCG beta-subunit was inactive as a gonadotropic agent to activate the adenylate cyclase in the rat testis. Intravitreal injection of rabbit luteinizing hormone, which was active as a gonadotropic agent, had no effect on IOP in 4 intravitreal concentrations ranging from 1 nM to 30 nM. These observations indicate that the mechanism of IOP decrease by intravitreal hCG is not related to its gonadotropic action. The IOP decrease in rabbits due to intravitreal hCG or its beta-subunit is probably related to a contaminant or an immune reaction.