Two classes of H pumps, H-K-ATPase and H-ATPase, contribute to luminal acidification and HCO3transport in the collecting duct (CD). At least two H-K-ATPase α-subunits are expressed in the CD: HKα1and HKα2. Both exhibit K dependence but have different inhibitor sensitivities. The HKα1H-K-ATPase is Sch-28080 sensitive, whereas the pharmacological profile of the HKα2H-K-ATPase is not completely understood. The present study used a nonpharmacological, genetic approach to determine the contribution of HKα1and HKα2to cortical CD (CCD) intercalated cell (IC) proton transport in mice fed a normal diet. Intracellular pH (pHi) recovery was determined in ICs using in vitro microperfusion of CCD after an acute intracellular acid load in wild-type mice and mice of the same strain lacking expression of HKα1, HKα2, or both H-K-ATPases (HKα1,2). A-type and B-type ICs were differentiated by luminal loading with BCECF-AM and peritubular chloride removal from CO2/HCO3-buffered solutions to identify the membrane locations of Cl/HCO3exchange activity. H-ATPase- and Na/H exchange-mediated H transport were inhibited with bafilomycin A1(100 nM) and EIPA (10 μM), respectively. Here, we report 1) initial pHiand buffering capacity were not significantly altered in the ICs of HKα-deficient mice, 2) either HKα1or HKα2deficiency resulted in slower acid extrusion, and 3) A-type ICs from HKα1,2-deficient mice had significantly slower acid extrusion compared with A-type ICs from HKα1-deficient mice alone. These studies are the first nonpharmacological demonstration that both HKα1and HKα2contribute to H secretion in both A-type and B-type ICs in animals fed a normal diet.