Inward Na⁺-HCOcotransport has previously been demonstrated in acidified duodenal epithelial cells, but the identity and localization of the mRNAs and proteins involved have not been determined. The molecular expression and localization of Na⁺-HCOcotransporters (NBCs) were studied by RT-PCR, sequence analysis, and immunohistochemistry. By fluorescence spectroscopy, the intracellular pH (pH_i) was recorded in suspensions of isolated murine duodenal epithelial cells loaded with 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. Proximal duodenal epithelial cells expressed mRNA encoding two electrogenic NBC1 isoforms and the electroneutral NBCn1. Both NBC1 and NBCn1 were localized to the basolateral membrane of proximal duodenal villus cells, whereas the crypt cells did not label with the anti-NBC antibodies. DIDS or removal of extracellular Cl⁻ increased pH_i, whereas an acidification was observed on removal of Na⁺ or both Na⁺ and Cl⁻. The effects of inhibitors and ionic dependence of acid/base transporters were consistent with both inward and outward Na⁺-HCO cotransport. Hence, we propose that NBCs are involved in both basolateral electroneutral HCO transport as well as basolateral electrogenic HCO transport in proximal duodenal villus cells.