E-selectin is an inducible adhesion molecule on endothelial cells. The internalization of this glycoprotein was investigated on tumor necrosis factor (TNF)-activated cultured human umbilical vein endothelial cells (HUVEC). Kinetics of intercellular adhesion molecule-1 (ICAM-1) were studied in parallel experiments. Internalization studies were performed with radioiodinated antibodies in an acid elution endocytosis assay, and by immunohistology; both approaches gave equivalent results. [125I]ENA1, a monoclonal antibody (mAb) specific for E-selectin, was internalized at a rate of approximately 1.7% of the membrane-bound [125I]mAb per minute. In contrast, less than 0.1% of membrane-bound [125I]RR1/1, an mAb specific for ICAM-1, was internalized per minute. TNF-activated HUVEC were immunostained and examined by light microscopy (LM) and electron microscopy (EM). LM revealed the presence of ENA1, but not RR1/1, after 30 minutes of incubation with these mAb in cytoplasmic vesicles, which were characterized as multivesicular bodies by EM. Without previous mAb exposure of the endothelial cells, both high amounts of E-selectin and bovine serum albumin complexed to colloidal gold, used as a marker for fluid-phase internalization, were detected in the same organelles, thus arguing against mAb interaction-induced E-selectin internalization. Furthermore, the amount of E-selectin surface expression was not influenced by ongoing mAb presence, also arguing against mAb interference with normal E-selectin kinetics. Taken together, these results indicate that TNF-activated HUVEC constitutively internalize E-selectin. Physiological significance of E-selectin internalization in the regulation of E-selectin membrane expression, and in clearing E-selectin ligands from the circulation, needs further investigation.