A procedure for the isolation and long-term in vitro cultivation of endothelial cells from rat cerebral cortical microvasculature is described. Migrating cells emerged from collagenase-treated microvessel fragments as early as 1 to 2 days in culture. Migration continued and marked proliferation began 5 to 7 days after isolation and continued up to 12 to 14 days. Cell colonies developed and consisted of endothelial cells as determined by phase contrast microscopy and cell culture behavior. Proliferation of the endothelial cells was significantly enhanced (3-to 4-fold) with the addition of endothelial cell growth supplement at a concentration of 150 microgram. per ml. Cultures with endothelial cell growth supplement (ECGS) retained their characteristic endothelial morphology for 6 to 8 weeks, after which they exhibited a gradual deterioration and loss of their phenotypic appearance. The endothelial origin of these cells was demonstrated by positive immunofluorescent staining for factor VIII antigen and angiotensin-converting enzyme and lack of binding of rat smooth muscle myosin antibody. Ultrastructural examination of confluent endothelial cell cultures revealed intercellular junctional complexes consisting of gap junctions, punctate fusions, and tight junctions. Since long-term endothelial cell cultures derived from the cerebral microvasculature retain characteristic endothelial cell markers and in vivo markers and in vivo features of brain capillary endothelium, they can serve as a useful model system to characterize microvascular endothelium in a variety of disease states.