The blood-brain barrier permeability of cyclosporin A (CsA), an immunosuppressive cyclic peptide, is restricted despite its highly lipophilic nature. In this study, the uptake of CsA by primary cultured bovine brain capillary endothelial cells (BCEC) was investigated in order to clarify whether P-glycoprotein (P-gp), an ATP-dependent drug efflux pump expressed in the luminal surface of BCEC, causes the decreased transport of CsA into the brain. P-gp, having a molecular mass of 130-140 kDa, was detected with anti-P-gp monoclonal antibody, C219, using western blot analysis of the membrane fraction isolated from the bovine brain capillary. The uptake of CsA by primary cultured bovine BCEC was time-dependent, and the steady-state uptake of CsA was increased in the presence of several multidrug resistance reversing agents including verapamil and steroid hormones and the substrate of P-gp in BCEC, vincristine. The steady-stage uptake was increased significantly to approximately 4-fold when cellular ATP was depleted by treating with 2,4-dinitrophenol, suggesting that the efflux process is ATP dependent. Furthermore, in the presence of an anti-P-gp monoclonal antibody, MRK16, at a 10 micrograms/mL concentration, the uptake of CsA was increased approximately 3-fold. These results suggest that the low permeability of CsA into the brain is caused by the active efflux from BCEC by P-gp present in the luminal surface of cells.