Bovine brain micro vessel endothelial cells have been isolated and grown in culture to monolayers. These endothelial cell monolayers have been characterized morphologically with electron microscopy, histochemically for brain endothelium enzyme markers, alkaline phosphatase and γ-glutamyl trans-peptidase, and by immunofluorescence to detect Factor VIII antigen, an exclusive endothelial antigen. Results of these studies indicate that the cells forming the monolayers are of endothelial origin and possess many features of the in vivo brain endothelium responsible for formation of the blood–brain barrier. This in vitro blood–brain barrier model system was used in experiments to determine the permeability of the cultured monolayer to sucrose, leucine, and propranolol. Leucine rapidly moved across the monolayers of this in vitro system and tended to plateau after approximately 10 min. In contrast, the rates of sucrose and propranolol movement were linear during a 1-hr observation period, with the rate of propranolol movement across the monolayer greater than that of sucrose. The ability to detect differences in the permeability of the monolayers to leucine, propranolol, and sucrose with radioactive tracers suggests that this in vitro model system will be an important tool for the investigation of the role of the blood–brain barrier in the delivery of centrally acting drugs and nutrients.