For many years, molecular interactions with vascular endothelium have been studied in vitro on cultured endothelial cells. Yet, it is clear that the different environmental conditions in vivo vs. in vitro may cause phenotypic drift and altered expression of cell surface molecules. In this study, we identify several endothelial surface proteins of similar apparent molecular mass by radioiodination of cultured microvascular cells and by intravascular radioiodination of rat heart endothelium in situ. The radioiodinated surface polypeptides detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (followed by autoradiography) were subjected to lectin affinity chromatography in order to provide an additional screen for identifying common surface glycoproteins and a means for partial characterization of their glycans. With a battery of 18 lectins, seven major (gp140, gp120, gp100, gp85, gp75, gp60, gp47) and 6 minor (gp330, gp300, gp180, gp160, gp150, gp42) glycoproteins were identified on the cultured cells each with a different lectin binding profile. The lectin binding profiles of many endothelial glycoproteins in situ were similar to those of their counterparts in culture. A common set of seven major glycoproteins with the same apparent molecular masses was found in situ as well as in vitro. These common glycoproteins were characterized further using both sialidase digestion and sequential lectin affinity chromatography of cell lysates. Most of the glycoproteins appear to have both complex-type N-linked and O-linked glycans except for gp60 with only O-linked glycans, gp47 with only complex N-linked sugars, and gp42 with only simple N-linked sugars. A subset of sialoglycoproteins (gp140, gp120, gp100, gp60, gp47) was identified. One of them, gp120, is podocalyxin based on immunoprecipitation with specific antiserum and another one, gp60, is a recently identified albumin binding protein on the surface of cultured microvascular endothelial cells. This study shows that gp60 is indeed present on the surface of endothelium in situ and that it is a sialoglycoprotein with typical O-linked glycans. It is apparent that the continuous type of microvascular endothelium can indeed express in culture and in situ a common set of major glycoproteins.