We have immunolocalized thrombomodulin (TM), (an endothelial cofactor protein for thrombin-mediated protein C activation), and its ligand thrombin (TH) by light- and electron microscopy on endothelial cells in culture and in situ. Our results show that: i) TM is expressed in small clusters on the basal and the apical surface of human umbilical vein endothelial cells (HUVEC), is absent from coated pits and coated vesicles, is internalized from the cell surface via plasmalemmal vesicles, and is associated with endosomes, including a tubulovesicular network within the cytoplasm; ii) exogenous TH binds at low surface density on HUVEC and colocalizes with TM in small TM clusters; iii) on the microvascular endothelium of heart, lung, and kidney, TM is expressed at high density on the luminal plasmalemma, is associated with a fraction of the plasmalemmal vesicle population; iv) and is found internalized in multivesicular bodies in TH perfused heart samples; v) in the same samples, perfused TH is detected at low surface density: on the luminal aspect of the endothelium within small TM clusters, in the introits and within plasmalemmal vesicles, in endosomal structures, on the abluminal plasmalemma, and within the pericapillary spaces. Our findings suggest that the high level of endothelial TM expression in situ provides a safety margin for random TH generation; they also demonstrate that bound TH is transcytosed across the endothelium by plasmalemmal vesicles which might represent an additional mechanism to remove TH from the circulation.