Tissue-specific gene expression within the brain capillary endothelium, which makes up the blood-brain barrier (BBB) in vivo, may lead to the production of brain capillary-specific proteins (BSPs). BSPs were defined in the present study by immunocytochemistry and Western blotting using a rabbit polyclonal antiserum made against a plasma membrane fraction of isolated bovine brain microvessels. Following absorption of the antiserum with acetone powders of rat liver and rat kidney, the antiserum illuminated only microvessels in brain and did not stain vessels in heart, choroid plexus, urinary bladder, liver, or kidney. Among these organs, the only other structure stained by the antiserum was the basolateral membrane of the bovine choroid plexus epithelium. Western blotting analysis showed that the antiserum reacted with a triplet of brain capillary proteins of 200, 53, and 45 kDa molecular weight. Although the 200-kDa protein was detected on Western immunoblot analysis with bovine choroid plexus, the 53- and 45-kDa proteins were specific to brain microvasculature. Immunocytochemical electron microscopic studies showed that the antiserum stained the plasma membrane of primary cultures of bovine capillary endothelium, but Western blot studies showed preservation of only the 200-kDa protein in primary tissue culture. These studies identify 53- and 45-kDa blood-brain barrier-specific proteins and show that the expression of these proteins is dependent on trophic factors not present in primary tissue culture. The future structural analyses of these proteins may lead to the elucidation of the possible function served in the regulation of BBB transport physiology.