P-glycoprotein (P-gp) is a highly-conserved membrane protein expressed in various multidrug-resistant cell lines. P-glycoprotein was detected in capillaries isolated from human, beef and rat brains with a Western immunoblotting procedure using the monoclonal antibody C219 (mAb C219) specific for P-gp. The mAb C219 detected a 180 kDa protein in brain capillaries isolated from all three species. The largest amount of antigen was detected in capillaries isolated from human brain. Specific binding was assessed by competitive inhibition of mAb C219 binding by the synthetic epitope VQEALD. The glycoprotein nature of the brain capillary proteins was confirmed by its sensitivity to N-glycanase treatment, which reduced their apparent molecular mass by 5 to 10 kDa. In addition, immunohistochemical studies using the antibodies C219, JSB-1 and C494 were performed. Bovine and rat capillaries showed reactivity only with the mAb C219. Heavy staining of human brain capillaries was observed with both antibodies C219 and JSB-1, while only weak staining was observed with antibody C494. These results clearly show that P-glycoprotein is strongly expressed at the blood-brain barrier (BBB) site and suggest that this protein may play a physiological role in regulating the access of certain molecules to the central nervous system, or in the secretory functions of the BBB.