In the intact primate eye in vivo, it may be difficult to determine whether an outflow facility response is due to direct effects on the trabecular meshwork or indirectly via an effect on ciliary muscle tone unless complicated surgical ciliary muscle disinsertion or isotope accumulation procedures are used. We established a monkey anterior segment organ culture system modeled after the human anterior segment organ culture system and determined its characteristics and response to H-7, an agent shown to increase trabecular outflow in vivo and in other organ culture systems. Outflow facility studies conducted using the monkey organ-cultured anterior segment system showed that: baseline values were comparable to those measured in vivo; washout occurred with time; the two-level constant pressure perfusion technique gave results comparable to the constant rate technique; species differences were found comparing baseline outflow facility and intraocular pressure in rhesus and cynomolgus monkey organ-cultured anterior segments; there was no effect of age on outflow facility in either species by one-way ANOVA; anterior segment exchange perfusion increased outflow facility if measured within 1 hr of the exchange; the magnitude of the response to H-7 was comparable to those reported in vivo and in other in vitro systems. The onset of the response to H-7 was delayed in most cases (day after H-7 exchange), compared to in vivo monkey studies (onset 6-9 min) and in vitro human and porcine organ culture systems (within 1 hr). Also the duration of the response (2-3 days from the onset) was longer than that found for in vitro human studies (1 day). Thus, the monkey organ-cultured anterior segment system can be used to determine the effects of pharmacological and biological agents on trabecular outflow.