We used video fluorescence microscopy of the vascular bed in the cremaster muscle of rat and mouse to study the transfer of plasmalemma vesicles (caveolae) across the microvessel barrier in situ. The water-soluble styryl pyridinium dye RH414, which adsorbs to and fluoresces at the membrane-water interface, was used as a marker for vesicular traffic through endothelial cells. Fluorescein isothiocyanate (FITC), similar in molecular size to the styryl pyridinium probe, was used to mark for dye transfer by the paracellular pathway. Transcellular dye flux was determined by comparing the fluorescence intensities of RH414 and FITC on either side of the vessel wall (i.e., in microvessel lumen and in muscle tissue at various distances from the microvessel wall). We observed that RH414 accumulated in the interstitium more rapidly than FITC. We next studied the role of the 60-kDa albumin-binding glycoprotein gp60, hypothesized to activate transcellular permeability, in stimulating the transcellular vesicle traffic. Introduction of anti-gp60 antibody into the microvessel to cross-link and activate gp60 markedly increased the transvascular flux of RH414. Control isotype-matched antibody had no effect on the RH414 flux. The sterol-binding agent filipin, which disassembles caveolae, inhibited the RH414 flux induced by gp60 cross-linking. The transfer of styryl pyridinium dyes in intact microvessels suggests that plasmalemmal membrane traffic across the skeletal muscle microvessel barrier is a constitutively active process. The results indicate that the gp60-dependent pathway is important in regulating endothelial permeability in situ via a transcellular mechanism.