The subcellular distribution in brain capillaries of alkaline phosphatase and Na+, K+-ATPase was investigated by two methods. Cytochemical studies using whole brain perfusion and electron microscopic examination indicated that alkaline phosphatase activity was located in both the luminal and antiluminal cytoplasmic membranes of the brain capillary endothelial cells. By contrast, the K+-dependent phosphatase activity associated with Na+, K+-ATPase was located in only the antiluminal membrane. Biochemical studies using membranes prepared by homogenization of isolated brain capillaries and density gradient centrifugation resulted in identification of two plasma membrane fractions. The light fraction contained alkaline phosphatase but very little Na+, K+-ATPase while the heavier fraction contained both enzyme activities. In addition, gamma-glutamyl transpeptidase showed a distribution similar to alkaline phosphatase while 5'-nucleotidase activity was distributed with the Na+, K+-ATPase activity. We conclude that the luminal and antiluminal membranes of brain capillaries are biochemically and functionally different. This polarity should permit active solute transport across brain capillary endothelial cells which are the cells responsible for the blood-brain barrier.