We present evidence that tissue distribution of two highly conserved Na⁺/H⁺exchanger isoforms, NHE2 and NHE4, differs significantly from previously published reports. Riboprobes unique to each of these antiporters, from 5′ (noncoding and coding) and 3′ coding regions, were used to analyze mRNA from adult rat kidney and intestine by ribonuclease protection assay and in situ hybridization. In contrast to earlier work that concluded that both NHE2 and NHE4 were expressed throughout the intestine and in the kidney, our data show that there is no NHE2 message in the kidney and NHE4 is not expressed in small or large intestine. Analyses of intestinal epithelial and kidney membrane proteins by an NHE2-specific antibody identified a doublet at <90 kDa in intestine but not in kidney. NHE2 is highly expressed in the Na⁺-absorptive epithelium of jejunum, ileum, and ascending and descending colon. NHE4 mRNA message is found in the inner medulla of the kidney as previously reported (C. Bookstein, M. W. Musch, A. DePaoli, Y. Xie, M. Villereal, M. C. Rao, and E. B. Chang. J. Biol. Chem. 269: 29704–29709, 1994) and not in the intestine. From these data, we speculate that neither NHE2 nor NHE4 has a role in renal Na⁺ absorption. NHE2 is likely involved in gut Na⁺ absorption, whereas NHE4 may have a specialized role in cell volume rectification of inner medullary collecting duct cells. Knowledge of the correct tissue and cell-specific distribution of these two antiporters should help significantly in understanding their physiological roles.