The purpose of this study was to compare human endothelial cells from Schlemm's canal (SCEs) and the trabecular meshwork (TMEs) in terms of ZO-1 isoform expression, hydraulic conductivity (HC) properties, and "giant" vacuole (GV) formation. The principal study methods were Western blot, RT-PCR, immunofluorescence, and perfusion chambers. Blot signals for alpha(+)- and alpha(-)-isoforms were similar in SCEs but less intense for the alpha(+)-relative to the alpha(-)-signal in TMEs. With the anti-alpha(+) antibody used at 1/50 dilution, binding occurred at cell borders of both cell types, but only to SCEs when used at a greater than or equal to 1/200 dilution in vitro and in vivo. SCEs were more resistive than TMEs (HC = 0.66 vs. 1.32 mul . min(-1) . mmHg(-1) . cm(-2); P < 0.001) when perfused from apex to base. When perfused in the other direction, SCEs were again more resistive (5.23 vs. 9.04 mu l center dot min(-1) center dot mmHg(-1) center dot cm(-2); P < 0.01). GV formation occurred only in SCEs as a function of flow direction, perfusion pressure, and time. We conclude that SCEs and TMEs have distinctive phenotypic properties involving their content of ZO-1 isoforms, barrier function, and GV formation.