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 --subunit (ng) is expressed in apical membranes of rodent prostate. Its highest level, as well as relative abundance, with respect to a1 isoform of Na,K-ATPase was observed in anterior lobe. Here, we have aimed to determine the subunit composition of nongastric H,K-ATPase, through the detailed analysis of the expression of all known X,K-ATPase -subunits in rat anterior prostate (AP). RT-PCR detects transcripts of -subunits of Na,K-ATPase only. Measurement of absolute protein content of these three -subunit isoforms using quantitative western blotting of AP membrane proteins indicate that the abundance order is 1>3>>2. Immunohistochemical experiments demonstrate that b1 is present predominantly in apical membranes, coinciding with ng, whereas b3 is localized in the basolateral compartment, coinciding with 1. This is the first direct demonstration of the ng-1 co-localization in situ indicating that, in rat AP, ang associates only with 1. The existence of ng1 complex has been confirmed by immunoprecipitation experiments. These results indicate that 1 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.