The physiological functions of the nongastric (colonic) H,K-ATPase (gene symbol Atp12a), unlike those of the Na,K-ATPase and gastric H,K-ATPase, are poorly understood. It has been suggested that it pumps Na⁺ more efficiently than H⁺; however, so far, there is no direct evidence that it pumps H⁺ in vivo. Previously, we found that the nongastric H,K-ATPase -subunit is expressed in apical membranes of rodent anterior prostate epithelium, in a complex with the Na,K-ATPase B1 subunit. Here we report the effects of Atp12a gene ablation on polarization of the B1 subunit and secretory function of the anterior prostate. In nongastric H,K-ATPase -deficient prostate, the Na,K-ATPase -subunit resided exclusively in basolateral membranes; however, the B1 subunit disappeared from apical membranes, demonstrating that B1 is an authentic subunit of the nongastric H,K-ATPase in vivo and that apical localization of B1 in the prostate is completely dependent on its association with the nongastric H,K-ATPase -subunit. A remarkable reduction in acidification of anterior prostate fluids was observed: pH 6.38 ± 0.14 for wild-type mice and 6.96 ± 0.10 for homozygous mutants. These results show that the nongastric H,K-ATPase is required for acidification of luminal prostate fluids, thereby providing a strong in vivo correlate of previous functional expression studies demonstrating that it operates as a proton pump.