PURPOSE: To determine the effect of Latrunculin (LAT)-A, a macrolide that binds to G-actin, which leads to the disassembly of actin filaments, on shape, junctions, and the cytoskeleton of cultured bovine aortic endothelial cells (BAECs) and on outflow facility in living monkeys. METHODS: Latrunculin-A dose-time-response relationships in BAECs were determined by immunofluorescence and phase contrast light microscopy, facility by two-level constant pressure anterior chamber perfusion. RESULTS: In BAECs, LAT-A caused dose- and incubation time- dependent destruction of actin bundles, cell separation, and cell loss. Cell-cell adhesions were more sensitive than focal contacts. Recovery was also dose- and time-dependent. In monkeys, exchange intracameral infusion and topical application of LAT-A induced dose- and time-dependent several-fold facility increases. The facility increase was completely reversed within several hours after drug removal. However, for at least 24 hours after a single topical LAT-A dose, perfusion with drug-free solution caused an accelerated increase in facility beyond that attributed to normal resistance washout. CONCLUSIONS: Pharmacological disorganization of the actin cytoskeleton in the trabecular meshwork by specific actin inhibitors like LAT-A may be a useful antiglaucoma strategy.