Na-H exchange (NHE) is one of the major non-nutritive Na absorptive pathways of the intestine and kidney. Of the four NHE isoforms that have been cloned, only one, NHE-3, appears to be epithelial specific. We have examined the regional and cellular expression of NHE-3 in the rat intestine. NHE-3 message in the small intestine was more abundant in the villus fractions of the small intestine than in the crypts. Analysis of NHE-3 mRNA distribution in the gut by in situ hybridization demonstrated epithelial cell specificity, as well as expression preferential to villus cells. NHE-1 message, in contrast, was ubiquitous, with slightly greater expression exhibited in the differentiating crypt and lower villus cells of the small intestine. Isoform-specific NHE-3 fusion protein antibody identified a 97-kD membrane protein in the upper villus cells of the small intestine, which was exclusively localized in the apical membrane. In contrast, antibody previously developed against the COOH-terminal region of human NHE-1 (McSwine, R. L., G. Babnigg, M. W. Musch, E. B. Chang, and M. L. Villereal, manuscript submitted for publication) identified a 110-kD basolateral membrane protein. These data suggest that unlike NHE-1, which probably serves a "housekeeping" function, NHE-3 may be involved in vectorial Na transport by the intestine.