A system was developed to measure the hydraulic conductivity of cultured monolayers of human trabecular meshwork (HTM) cells. By optimizing the cell growth conditions and evaluating a number of filter supports, confluent HTM cells in single layers were obtained for measurement of hydraulic conductivity. The HTM monolayers had hydraulic conductivities of 0.3-2.0 microliters/min/mm Hg/cm2 measured at near-physiological flow rates. Evaluations of cytochalasin B (CB) effects on the hydraulic conductivity of our HTM monolayers revealed that CB (10(-6) to 10(-5) M) caused a dramatic dose-related increase in conductivity within 10 to 30 min, which parallels CB effects on outflow facility in vivo. Morphologic observations show that the increase in hydraulic conductivity was accompanied by a retraction of the trabecular cells and widening of the intercellular spaces. Our findings suggest that growth of HTM cells on filter supports can provide a useful in vitro system to study the regulation of aqueous outflow.