E-selectin is a cytokine-inducible membrane glycoprotein capable of mediating adhesion of leukocytes to endothelial cells. It is highly glycosylated, containing 11 sites for N-linked glycosylation. N-Glycosylation of E-selectin was analyzed by endoglycosidase treatment. Analysis of immunoprecipitated E-selectin from human umbilical vein endothelial cells (HUVEC) by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate showed that E-selectin was completely resistant to endoglycosidase H, but sensitive to peptide N-glycanase F digestion. This suggested that all N-linked oligosaccharide chains were of the complex type. The role of N-linked glycosylation in surface expression and secretion of E-selectin was studied using interleukin-1-stimulated HUVEC, cultured in the presence of the soluble glycosylation inhibitors tunicamycin or castanospermine. Cell surface expression was analyzed by indirect flow cytometry. N-Glycosylation was blocked by tunicamycin, and resulted in a significantly reduced surface expression of E-selectin, whereas castanospermine only marginally reduced E-selectin expression. The deglycosylated forms of E-selectin were also found to be fully capable of mediating adhesion of HT-29 cells in vitro. In conclusion, these studies show that E-selectin is heavily glycosylated with complex type N-linked oligosaccharides and that N-glycosylation is important for expression of E-selectin on human endothelial cells.