The cortical collecting duct (CCD) B cell possesses an apical anion exchanger dissimilar to AE1, AE2, and AE3. The purpose of these studies was to characterize this transporter more fully by examining its regulation by CO2 and HCO3. We measured intracellular pH (pHi) in single intercalated cells of in vitro microperfused CCD using the fluorescent, pH-sensitive dye, 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). In the absence of extracellular CO2/HCO3, luminal Cl removal caused reversible intracellular alkalinization, identifying this transporter as a Cl/base exchanger able to transport bases other than HCO3. Adding extracellular CO2/HCO3 decreased B cell pHi while simultaneously increasing Cl/base exchange activity. Since intracellular acidification inhibits AE1, AE2, and AE3, we examined mechanisms other than pHi by which the stimulation occurred. These studies showed that B cell apical anion exchange activity was CO2 stimulated and carbonic anhydrase dependent. Moreover, the stimulation was independent of luminal bicarbonate, luminal pH or pHi, and changes in buffer capacity. We conclude that the B cell possesses an apical Cl/base exchanger whose activity is regulated by CO2-stimulated, carbonic anhydrase-dependent cytoplasmic HCO3 formation.