Various vesicular carriers transport select molecular cargo between intracellular compartments utilizing a budding mechanism with docking and fusion of individual vesicles with their target membranes. This fusion requires key intracellular component(s) that are sensitive to alkylation with N-ethylmaleimide (NEM). In endothelium, caveolae may mediate endocytosis and transcytosis of select macromolecules. If caveolae utilize a mechanism similar to other vesicular carriers, then their transport should also be sensitive to NEM. The following tracers were chosen based on their pathway specificity: 1) albumin-gold complexes (A-Au) that bind gp30 and gp18 for endocytosis by caveolae, 2) native albumin that binds albondin and is transported by caveolae, 3) ferritin as a fluid-phase probe transcytosed by caveolae, and 4) inulin as a paracellular probe. In culture, NEM significantly inhibited A-Au uptake and delivery to endosomes but not endothelial cell surface binding. In rat lung, NEM reduced capillary permeability to albumin and the tissue uptake of ferritin and A-Au, but not inulin, indicating inhibition of caveolae-mediated but not paracellular transport. Neutral but not charged alkylating agents inhibited A-Au uptake, consistent with their relative abilities to cross membranes and modify intracellular factors. Like other vesicular pathways, endothelial caveolae transport their select ligands utilizing a NEM-sensitive mechanism, apparently requiring vesicle-membrane fusion.