Albumin reduces capillary permeability and acts as a carrier for various small molecules. Recently, we identified a 60-kDa sialoglycoprotein (gp60) on the surface of cultured rat microvascular endothelial cells (MEC) that binds albumin and antiglycophorin serum (alpha-gp). We verified that alpha-gp recognizes the albumin-binding gp60 by affinity, purifying proteins from MEC extracts using immobilized albumin. gp60 was immunoblotted with alpha-gp only when the MEC extract was reacted with albumin and not in controls. We immunoprecipitated gp60 from biosynthetically radiolabeled MEC lysates and from extracts containing endothelial surface proteins of isolated rat hearts that were radioiodinated in situ. gp60 was immunoblotted selectively in rat tissue microvascular beds lined with continuous endothelium (heart, lung, diaphragm, fat, skeletal muscle, mesentery, and duodenal muscularis but not cortical brain) and not those exclusively lined with fenestrated or sinusoidal endothelium (adrenal, pancreas, liver, and small intestinal mucosa). MEC isolated from rat heart, lung, and epididymal fat pad expressed gp60 and bound albumin, whereas various nonendothelial cells and brain-derived MEC did not. gp60 is an albumin-binding glycoprotein expressed specifically on the surface of continuous endothelium that binds albumin apparently not only to initiate its transcytosis via plasmalemmal vesicles but also to increase capillary permselectivity.