The presence of P-glycoprotein in the cell plasma membrane limits the penetration of many cytotoxic substances into cells that express the gene product. There is considerable evidence also to indicate that Pglycoprotein is expressed as part of the normal bloodbrain barrier in the luminal membranes of the cerebral capillary endothelial cells, where it presumably performs a protective function for the brain. This report describes the functional expression of P-glycoprotein in an immortalised cell line, RBE4, derived from rat cerebral capillary endothelial cells. The expression of P-glycoprotein is demonstrated by western immunoblotting and by immunogold and fluorescent staining with monoclonal antibodies. The cellular accumulation of [ 3H]colchicineand [3H]vinblastine is investigated and shown to be enhanced by the presence of azidothymidine, chlorpromazine, verapamil, cyclosporin A, and PSC 833 ([3'-keto-Bmt1]-[Val2]-cyclosporin) at 50 or 100 1iM concentration. It is concluded that the RBE4 cell line is a valuable tool for investigating the mechanisms of P-glycoprotein activity both in the blood-brain barrier and in multidrug resistance in general. Key Words: P-glycoprotein-Multidrug resistance-mdr reversal -Bloodbrain barrier-RBE4 cells -Vincristine -Vinblastine -Azidothymidine -Chlorpromazine-Verapamil-Cyclosporin A-PSC 833.