The physiological functions of nongastric (colonic) H-K-ATPase (gene symbol Atp12a), unlike those of 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 α-subunit is expressed in apical membranes of rodent anterior prostate epithelium, in a complex with the Na-K-ATPase β1-subunit. Here we report the effects of Atp12a gene ablation on polarization of the β1-subunit and secretory function of the anterior prostate. In nongastric H-K-ATPase-deficient prostate, the Na-K-ATPase α-subunit resided exclusively in basolateral membranes; however, the β1-subunit disappeared from apical membranes, demonstrating that β1is an authentic subunit of nongastric H-K-ATPase in vivo and that apical localization of β1in the prostate is completely dependent on its association with the nongastric H-K-ATPase α-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 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.