Basolateral membranes of turtle (Pseudemys scripta) colon epithelial cells exhibit robust Na+/H+ exchange activity that can be activated by cell shrinkage and is blocked by amiloride [M. A. Post and D. C. Dawson. Am. J. Physiol. 262 (Cell Physiol. 31): C1089-C1094, 1992]. The colonic epithelium actively absorbs Na+ and secretes K+ and HCO3, but the role of basolateral Na+/H+ exchange, if any, in transepithelial transport is unknown. The current studies were undertaken to identify the gene product(s) responsible for the observed basolateral Na+/H+ exchange activity and to determine the cellular localization of the reptilian Na+/H+ exchange protein. We cloned and sequenced partial-length cDNAs that are likely to encode a reptilian homologue of the mammalian NHE-1 Na+/H+ exchanger isoform. The partial- length cDNAs were >80% identical to mammalian NHE-1 homologues at the nucleotide level and recognized a transcript (~5.8-6.0 kb) in RNA isolated from turtle colon, small intestine, stomach, kidney, urinary bladder, heart, and liver. In situ hybridization showed that mRNA encoding the reptile homologue of NHE-1 was expressed predominantly in the epithelial cells of these tissues. Immunofluorescent localization of the reptilian Na+/H+ exchanger protein using an antibody raised against a human NHE-1 fusion protein confirmed that protein expression paralleled abundant mRNA expression in epithelial cells of turtle stomach and colon, as well as in some nephron segments, and showed that the reptile NHE-1 homologue was localized exclusively to the basolateral membranes of these cells. The relatively high level of NHE-1 expression in epithelial cells, particularly those of the colon and stomach, suggests that NHE-1 function is important for the maintenance or regulation of ion transport processes that occur in these cell types.