Prostaglandin F 2x (PGF 2x) is a powerful ocular hypotensive agent in rabbit, cat, dog, monkey and human. In cynomolgus monkeys, the intraocular pressure (IOP) lowering is due to increased uveoscleral outflow (F u,). Because the anatomy of the rabbit outflorv apparatus differs significantly from that of the primate, we sought to determine whether the mechanism of the PGF 2x-induced IOP fall was the same. PGF 2x tromethamine salt (PGF 2x-TS) (50 μg) applied to one eye of 14 conscious rabbits produced a significant IOP fall of 7.4±0.9 mmHg (P < 0.001). In untreated control eyes. F u, determined from the quantity of intracamerally perfused [ 125I]albumin found in the ocular and periocular tissues accounted for 5 −8 of total aqueous outflow. In 15 unilaterally PGF 2x-treated rabbits, after 4-6 hr dosing F u, was 49±14% higher in the treated than in the contralateral control eyes. Total outflow facility of outflow from the anterior chamber to the general circulation were measured concurrently in 11 rabbits using a two-level constant pressure perfusion and isotope accumulation technique. Both facilities tended to be higher in the treated eyes than in the controls, with a strong correlation between drug-induced changes in total facility and changes in facility of flow to blood (r = 0.85, P < 0.001). In eight rabbits treated unilaterally with 50μg PGF 2x-TS, the fluorophotometrically determined aqueous formation rate was probably not decreased relative to control eyes. Protein levels in the aqueous humor were approximately eight-fold higher in PG-treated vs. control eyes, suggesting a drug-induced compromise of the blood-aqueous barrier. The hypotensive mechanism of PGF 2x in the rabbit differs from that of the primate, perhaps due to significant differences in the outflow and orbital anatomy.