Cultures of endothelial cells (EC) derived from human (autopsy) and canine brain microvessels were characterized with respect to growth, morphology, and biochemical features. The endothelial nature of these cells was confirmed by analyses of angiotensin-converting enzyme activity, Factor VIII-related antigen, and ultrastructure. Human EC required coated substrates and tumor-conditioned medium to achieve rapid growth, and cells derived from human microvessels were morphologically diverse. In contrast, canine EC grew rapidly on plastic substrates and produced colonies of uniform morphology. Morphological variations of EC were associated with the use of heparin-containing medium and with the use of a commercially-prepared basement membrane extract (Matrigel). Lectin histochemistry demonstrated that human EC lack the abundant alpha-galactose residues characteristic of canine EC membranes and organelles and that canine EC lack the alpha-N-acetylgalactosamine residues which are associated with human EC. The lectin Ricinus communis agglutinin I may be useful for distinguishing canine EC from pericytes. Gel electrophoresis of membrane proteins revealed protein bands present in human EC at Mr 210,000 and 37,000-39,500 which were not present in canine EC. These proteins may be related to the presence of junctional complexes in cultures of human EC.