Primary cultures of human cerebral endothelial cells were established from microvessels isolated from cortical fragments removed at surgery for seizure disorder and from brains at autopsy. A uniform population of cells growing in close association to each other formed confluent monolayers by 7 to 10 days in culture. They contained factor VIII/Von Willebrand antigen, the most specific marker for cells of endothelial origin, and showed lectin-binding sites for Ulex europaeus agglutinin characteristic of human endothelium. Cultured cells formed thin, continuous monolayers, contained few pinocytotic vesicles, and were joined together by tight junctional complexes. More than 99% of the intercellular junctions restricted the transendothelial passage of horseradish peroxidase. Monolayers of human brain microvessel endothelial cells thus resemble cerebral endothelium in vivo and should provide a useful in vitro model for studies of the biology of these cells and their role in the pathogenesis of certain human central nervous system diseases associated with abnormal blood-brain barrier function.