Cl-/HCO3- exchange is present in all three cell types of the rabbit cortical collecting tubule, yet may mediate a different function in each cell type. The purpose of this study was to characterize further the location, function, and regulation of Cl-/HCO3- exchange in two cell types using measurements of intracellular pH (pHi). In the principal cell there was no evidence for apical Cl-/HCO3- exchange, including no change in pHi with increases in luminal HCO3-. The principal cell possesses a basolateral Cl-/HCO3- exchanger that is inactive normally but stimulated by intracellular alkalosis. Decreased PCO2 results in increased pHi associated with activation of Cl-/HCO3- exchange and partial recovery of pHi. In contrast, the beta-intercalated cell possesses an apical Cl-/HCO3- exchanger and alkalinizes with increases in luminal HCO3-. Also in contrast to the principal cell, the beta-intercalated cell apical Cl-/HCO3- exchanger does not appear to be involved in pHi regulation and may be specifically modified for transcellular HCO3- transport. In conclusion, the separate Cl-/HCO3- exchangers in the principal cell and the beta-intercalated cell not only have opposite polarity but are regulated differently.