The renal cortical collecting duct (CCD) plays an important role in systemic acid-base homeostasis. The β-intercalated cells secrete most of the HCO, which is mediated by a luminal, DIDS-insensitive, Cl⁻/HCO exchange. The identity of the luminal exchanger is a matter of debate. Anion exchanger isoform 4 (AE4) cloned from the rabbit kidney was proposed to perform this function (Tsuganezawa H et al. J Biol Chem 276: 8180–8189, 2001). By contrast, it was proposed (Royaux IE et al.Proc Natl Acad Sci USA 98: 4221–4226, 2001) that pendrin accomplishes this function in the mouse CCD. In the present work, we cloned, localized, and characterized the function of the rat AE4. Northern blot and RT-PCR showed high levels of AE4 mRNA in the CCD. Expression in HEK-293 and LLC-PK₁ cells showed that AE4 is targeted to the plasma membrane. Measurement of intracellular pH (pH_i) revealed that AE4 indeed functions as a Cl⁻/HCO exchanger. However, AE4 activity was inhibited by DIDS. Immunolocalization revealed species-specific expression of AE4. In the rat and mouse CCD and the mouse SMG duct AE4 was in the basolateral membrane. By contrast, in the rabbit, AE4 was in the luminal and lateral membranes. In both, the rat and rabbit CCD AE4 was in α-intercalated cells. Importantly, localization of AE4 was not affected by the systemic acid-base status of the rats. Therefore, we conclude that expression and possibly function of AE4 is species specific. In the rat and mouse AE4 functions as a Cl⁻/HCO exchanger in the basolateral membrane of α-intercalated cells and may participate in HCO absorption. In the rabbit AE4 may contribute to HCO secretion.