The structural organization of nongastric H-K-ATPase, unlike that of closely related Na-K-ATPase and gastric H-K-ATPase, is not well characterized. Recently, we demonstrated that nongastric H-K-ATPase α-subunit (α_ng) is expressed in apical membranes of rodent prostate. Its highest level, as well as relative abundance, with respect to α₁-isoform of Na-K-ATPase, was observed in anterior lobe. Here, we aimed to determine the subunit composition of nongastric H-K-ATPase through the detailed analysis of the expression of all known X-K-ATPase β-subunits in rat anterior prostate (AP). RT-PCR detects transcripts of β-subunits of Na-K-ATPase only. Measurement of absolute protein content of these three β-subunit isoforms, with the use of quantitative Western blotting of AP membrane proteins, indicates that the abundance order is β₁ > β₃ ≫ β₂. Immunohistochemical experiments demonstrate that β₁ is present predominantly in apical membranes, coinciding with α_ng, whereas β₃ is localized in the basolateral compartment, coinciding with α₁. This is the first direct demonstration of the α_ng-β₁ colocalization in situ indicating that, in rat AP, α_ng associates only with β₁. The existence of α_ng-β₁ complex has been confirmed by immunoprecipitation experiments. These results indicate that β₁-isoform functions as the authentic subunit of Na-K-ATPase and nongastric H-K-ATPase. Putatively, the intracellular polarization of X-K-ATPase isoforms depends on interaction with other proteins.