Purpose. To determine in normotensive cynomolgus monkeys, the effects of topical 8-iso prostaglandin (PG)E2 on intraocular pressure (IOP), aqueous humor formation (AHF), uveoscleral outflow (Fu), and total and trabecular outflow facility. methods. IOP was measured by Goldmann applanation tonometry under ketamine anesthesia after single or twice-daily topical treatments with 8-iso PGE2. With animals under pentobarbital anesthesia, AHF and flow to blood (equated to trabecular outflow) were determined by anterior chamber perfusion with radioactively labeled albumin solution. Fu and trabecular outflow facility were calculated from these measurements. Total outflow facility was measured by two-level, constant-pressure perfusion. results. IOP was not significantly changed after single or multiple 10-μg doses of 8-iso PGE2. The 25-μg dose significantly decreased IOP by 2 to 3 mm Hg compared to the contralateral vehicle-treated control 4 to 6 hours after a single dose and by 3 to 5 mm Hg within 1.5 hours after twice-daily treatments for 4 to 5 days. Total outflow facility corrected for control eye washout was increased by an apparent 37% (P < 0.02, n = 7) from 2 to 3.5 hours after the ninth dose, largely due to outlier values obtained in one monkey. Isotope studies performed after twice-daily treatments totaling 9 to 29 doses showed no change in AHF, trabecular outflow facility, or total outflow facility. Relative to AHF, trabecular outflow was significantly decreased, and the calculated Fu was significantly increased when all data were analyzed. conclusions. The present findings are consistent with lowering of IOP by 8-iso PGE2, primarily by increasing Fu. A direct effect on the trabecular meshwork was not indicated by these in vivo studies.