Intrahepatic bile duct cells (cholangiocytes) play an important role in the secretion and alkalinization of bile. Both Na⁺/H⁺ exchange (NHE) and Cl/HCO₃ exchange (AE) contribute to these functions, but their functional distribution between the apical and basolateral membrane domains remains speculative. We have addressed this issue in a normal rat cholangiocyte cell line (NRC-1), which maintains a polarized distribution of membrane markers. Gene expression of AE and NHE isoforms was studied by RT-PCR. For functional studies, cells were placed in a chamber that allowed separate perfusion of the apical and basolateral aspect of the epithelial sheet; intracellular pH (pH_i) was measured by 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein microfluorometry. In HCO₃-CO₂free medium and in the presence of apical amiloride, pH_i recovery from an acid load was Na⁺ dependent and was inhibited by basolateral amiloride and by HOE-642 (10 µM), consistent with basolateral localization of the NHE1 isoform, which had clearly expressed mRNA. Apical Na⁺ readmission induced a slow pH_i recovery that was inhibited by apical administration of 1 mM HOE-642 or amiloride. Among the apical NHE isoforms, NHE2 but not NHE3 gene expression was detected. The AE1 gene was not expressed, but two different variants of AE2 mRNAs (AE2a and AE2b) were detected; pH_i experiments disclosed AE activities at both sides of the membrane, but only apical AE was activated by cAMP. In conclusion, these studies provide the first functional description of acid-base transporters in a polarized cholangiocyte cell line. NHE1, NHE2, AE2a, and AE2b isoforms are expressed and show different membrane polarity, functional properties, and sensitivity to inhibitors. These observations add a considerable level of complexity to current models of electrolyte transport in cholangiocytes.