Previous studies have shown that the inhibition of Rho kinase is involved in the regulation of outflow facility in the live rabbit eye and the enucleated porcine eye. However, it is unknown whether the Rho kinase inhibition will do the same in non-human primates. To determine if the Rho kinase inhibitor Y-27632 will reduce outflow resistance in the live monkey eye, if Y-27632 and the phosphatase inhibitor calyculin A (Caly-A which antagonises Y-27632-induced MLC dephosphorylation) will affect outflow facility differently, and if the latter will inhibit effect of the former on facility, we studied effects of Y-27632 and Caly-A on outflow facility in living monkeys separately and concurrently. Total outflow facility was measured by 2-level constant pressure perfusion of the anterior chamber (AC) before and after exchange with different doses of Y-27632 (1, 10 and 100 microM) or Caly-A (10, 50 and 100 nM), or vehicles, followed by continuous AC infusion of corresponding drug/vehicle solution, in opposite eyes of cynomolgus or rhesus monkeys. The effect of 100 microM Y-27632 or 100 nM Caly-A vs vehicle and the effect of 100 microM Y-27632+100 nM Caly-A vs 100 microM Y-27632 alone on outflow facility were also determined in monkeys pre-treated topically with 10 microl of 1% atropine in both eyes 1 hr before perfusion. Both Y-27632 and Caly-A dose-dependently increased outflow facility by up to 2-3 fold in monkeys, adjusted for baseline and contralateral control eye washout. Pre-treatment with 1% topical atropine partially inhibited the effect of 100 nM Caly-A, but not 100 microM Y-27632, on outflow facility. 100 nM Caly-A gradually and partially inhibited the Y-27632-induced facility increase. In conclusion, Y-27632 increases outflow facility in monkeys presumably by inhibiting cellular contractility in the TM. Caly-A increases outflow facility by complicated mechanisms perhaps including drug-induced ciliary muscle contraction and cytoskeletal reorganisation in TM cells. The partial inhibitory effect of Caly-A on the Y-27632-induced increase in outflow facility may reflect the former partially inhibiting the latter's relaxation of cells in the TM.