Cell culture models have been extensively used for studies of blood-brain barrier (BBB) function. However, several in vitro models fail to reproduce some, if not most, of the physiological and morphological properties of in situ brain microvascular endothelial cells. We have recently developed a dynamic, tridimensional BBB model where endothelial cells exposed to intraluminal flow form a barrier to ions and proteins following prolonged co-culturing with glia. We have further characterized this cell culture model to determine whether these barrier properties were due to expression of a BBB phenotype. Endothelial cells of human, bovine or rodent origin were used. When co-cultured with glia, intraluminally grown endothelial cells developed features similar to in vivo endothelial cells, including tight junctional contacts at interdigitating processes and a high transendothelial resistance. This in vitro BBB was characterized by the expression of an abluminal, ouabain-sensitive Na/K pump, and thus favored passage of potassium ions towards the lumen while preventing K+ extravasation. Similarly, the in vitro BBB prevented the passage of blood-brain barrier-impermeant drugs (such as morphine, sucrose and mannitol) while allowing extraluminal accumulation of lipophylic substances such as theophylline. Finally, expression of stereo-selective transporters for Aspartate was revealed by tracer studies. We conclude that the in vitro dynamic BBB model may become an useful tool for the studies of BBB-function and for the testing of drug passage across the brain endothelial monolayer.