The hypothesis that P‐glycoprotein plays a functional role at the brain capillary endothelium, which makes up the blood‐brain barrier in vivo, is based largely on immunocytochemical studies showing immunoreactive P‐glycoprotein localized to either isolated brain microvessels or microvessels within tissue sections. The present studies use the MRK16 monoclonal antibody to human P‐glycoprotein to demonstrate that the pattern of immunolocalization of P‐glycoprotein in microvessels of human or primate brain is similar to the pattern of immunolocalization of an astrocyte protein, glial fibrillary acidic protein. In contrast, the discontinuous staining pattern of MRK16 is not colocalized with the continuous immunostaining of the brain endothelial GLUT1 glucose transporter. The MRK16 antibody was radiolabeled with [125I]‐iodine, and 125I‐MRK16 avidly bound isolated human brain capillaries via a saturable mechanism. However, the 125I‐MRK16 antibody was not taken up by primate brain capillaries in vivo following intravenous injection. In conclusion, these studies provide evidence that P‐glycoprotein does not play a functional role at the luminal membrane of the brain capillary endothelium in vivo, and that a principal site of immunoreactive P‐glycoprotein in brain microvasculature is localized to astrocyte foot processes.