The identity of Na+/H+ exchanger (NHE) isoforms in the human small intestine and colon and their role in vectorial Na+ absorption are not known. The present studies were undertaken to examine the regional and vertical axis distribution of NHE-1, NHE-2, and NHE-3 mRNA in the human intestine. Ribonuclease protection assays were used to quantitate the levels of mRNA of these isoforms in various regions of the human intestine. In situ hybridization technique was used to localize NHE-2 and NHE-3 mRNA in the colon. The NHE-1 isoform message was present uniformly throughout the length of the human intestine. In contrast, mRNA levels for human NHE-2 and NHE-3 isoforms demonstrated significant regional differences. The NHE-3 abundance was found in decreasing order: ileum > jejunum > proximal colon = distal colon. The NHE-2 message level in the distal colon was significantly higher than in the proximal colon but was evenly distributed in the small intestine. In addition, NHE-2 mRNA was present in surface epithelial cells as well as in cells of the crypt region, suggesting the presence of NHE-2 message throughout the vertical axis of the colonic crypts. In contrast, NHE-3 mRNA was localized to surface colonocytes in the proximal colon. On the basis of this tissue-specific localization of NHE-2 and NHE-3 mRNA, it can be speculated that the relative contribution of NHE-2 and NHE-3 isoforms in Na+ absorption in the human intestine may be region specific, and these putative apical isoforms may be differentially regulated.