The properties of 125I-insulin binding were assessed in endothelial cells prepared from the veins and the arteries of human umbilical cords. The endothelial nature of both the arterial and venous cultures was documented by the presence of characteristic endothelial features, including Weibel-Palade bodies, factor VIII antigen, and morphology. Both arterial and venous cells possessed typical receptors for insulin on the basis of specificity of binding, curvilinear Scatchard plots, affinity profiles, pH dependency, and dissociation kinetics. Arterial cells bound at least 2.5 times more insulin than did venous cells, whether studied at 4 h, 24 h, or 72 h after in vitro plating. We conclude that (1) specific receptors for insulin are present on human arterial as well as human venous endothelial cells and (2) the concentration of insulin receptors varies among endothelial cells derived from different vascular sources.