We generated monoclonal antibodies (MAbs) against cultured bovine brain microvessel endothelial cells (BMEC) for use as probes to study membrane protein traffic and polarity. One MAb recognized a heterogeneous family of acidic sulfoglycoproteins called gp4A4 with molecular weights of 50-65 KD and 85 KD. Gp4A4 is a long-lived integral membrane protein which resides mostly at the plasma membrane, and a portion appears to be in equilibrium with an intracellular pool via endocytosis. Gp4A4 is expressed by many endothelial cells, except for fenestrated capillaries in choroid plexus, and specific epithelial cells in bile duct, kidney, and choroid plexus. A comparison of indirect immunoperoxidase and immunofluorescence detection using semi-thin cryosections gave contrasting results on the apparent distribution of gp4A4 on the apical and basolateral membranes of cerebral endothelia and choroid plexus epithelia. Immunogold labeling of ultra-thin cryosections showed that gp4A4 was expressed by the apical and basolateral membrane domains of BMEC and choroid plexus epithelia. This was consistent with the results using indirect immunofluorescence microscopy. On an average, gp4A4 expression by cerebral endothelia was not asymmetric and was considerably variable between capillaries. These results emphasize the need to compare several different techniques in assessing polarized expression of cell surface antigens in vivo.