Polyclonal antibodies were raised to peptides selected from three different regions of the cloned rabbit intestinal Na+/glucose cotransporter. Western blot analysis was used to identify the fully mature protein in intestinal and renal brush borders. Two of the antibodies specifically identified a approximately 70-kDa protein band in rabbit intestinal brush borders but did not specifically immunoreact with membranes that do not have Na(+)-dependent glucose transport activity. The immunoreactive proteins had an apparent isoelectric point between pH 4.7 and 6.8. The antibodies also specifically recognized a similar-sized protein in human and seven other mammalian intestinal brush borders. Similar protein bands were identified in four mammalian renal brush-border membranes, indicating shared epitopes between intestinal and renal cotransport proteins. In some species, e.g., lamb and pig, the epitope for one antibody was missing in both intestinal and renal brush borders, suggesting that this epitope is not essential for function. These results suggest that 1) the cloned intestinal Na+/glucose cotransporter is that identified in earlier biochemical studies, 2) there is close structural similarity between intestinal and renal cotransporters, and 3) the structure of these proteins has been conserved during evolution.