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To determine the mechanism of H-7-induced outflow resistance decrease, the reversibility of H-7 effects on outflow pathway was studied physiologically and morphologically in live monkey eyes. Total outflow facility was measured by two-level constant pressure perfusion before (baseline measurement) and after (post-drug measurement) anterior chamber (AC) exchange with 300 μm H-7 or vehicle in opposite eyes of eight monkeys. H-7 was then removed by AC exchange with drug-free vehicle in both eyes, followed by a 2·5 hr waiting period, after which outflow facility was measured again with (Group 2; n=4) or without (Group 1; n=4) another preceding drug-free AC exchange. For morphological study, five monkeys were initially perfused similarly to Group 1 in physiology, but the facility measurement beginning 2·5 hr after drug removal was either omitted or replaced by gold solution infusion. Following baseline measurement, two of the five monkeys received H-7 or vehicle in opposite eyes, while three monkeys received H-7 in both eyes 2·5 hr apart, contributing one H-7-treated ‘recovery’ eye and one H-7-treated ‘acute’ eye. After perfusion, both eyes of all five monkeys were studied by light and electron microscopy. Outflow facility during post-drug measurement in the H-7-treated eye was significantly increased by two-fold. However, the facility increase was reduced when measured beginning 2·5 hr after drug removal, with the reduction being greater in Group 1. ‘Recovered’ outflow facility after drug removal gradually increased again under continuous AC infusion with drug-free vehicle. Morphologically, major changes in and around Schlemm's canal (SC) in the H-7-treated ‘acute’ eye included protrusion of the entire inner wall (IW) into SC, relaxation of the IW cells and reorganization of the IW cytoskeleton. The changes in IW cells and juxtacanalicular region of the H-7-treated ‘recovery’ eye were non-uniform, with areas resembling the vehicle-treated eye (‘contracted areas’) and areas resembling the H-7-treated ‘acute’ eye (‘relaxed areas’). The average junction-to-junction distances in the IW cells of the H-7-treated ‘recovery’ eye were intermediate between the vehicle-treated eye and the H-7-treated ‘acute’ eye. In conclusion, H-7’s effect on outflow facility seems reversible, but AC exchange or continuous infusion with drug-free vehicle can re-elevate the ‘recovered’ outflow facility. Major morphological changes in the TM immediately after H-7 include IW protrusion, cellular relaxation and cytoskeleton reorganization. The decrease in ‘relaxed areas’ in the TM, in conjunction with the reversed outflow facility, 2·5 hr after drug removal suggests that cellular relaxation in the TM is the structural basis for H-7-induced increase in outflow facility.