Two classes of H pumps, H,K-ATPase and H-ATPase, contribute to luminal acidification and HCO₃ secretion in the collecting duct (CD). At least two H,K-ATPase α-subunits are expressed in the CD: HKα₁ and HKα₂. Both exhibit K-dependence but have different inhibitor sensitivities. The HKα₁ H,K-ATPase is Sch-28080-sensitive whereas the pharmacological profile of the HKα₂ H,K-ATPase is not completely understood. The present study used a non-pharmacological, genetic approach to determine the contribution of HKα₁ and HKα₂ to cortical CD (CCD) intercalated cell (IC) proton transport in mice fed a normal diet. Intracellular pH (pH_i) recovery was determined in ICs using in vitro microperfusion of CCD after an acute intracellular acid load in wild-type (WT) mice and mice of the same strain lacking expression of HKα₁, HKα₂2, or both H,K-ATPases (HKα_1,2). 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/HCO₃ exchange activity. H-ATPase- and Na/H exchange-mediated H transport were inhibited with bafilomycin A₁ (100 nM) and ethyl-isopropyl-amiloride (EIPA 10 μM), respectively. Here we report: (1) initial pH_i and buffering capacity were not significantly altered in the ICs of HKα deficient mice; (2) either HKα₁ or HKα₂ deficiency resulted in slower acid extrusion in ICs; and (3) A-type ICs from HKα_1,2 deficient mice had significantly slower acid extrusion when compared to the A-type ICs from HKα₁ deficient mice alone.