Short-term aldosterone coordinately regulates the cell-surface expression of luminal epithelial sodium channels (ENaC) and of basolateral Na(+) pumps (Na(+), K(+)-ATPase alpha1-beta1) in aldosterone-sensitive distal nephron (ASDN) cells. To address the question of whether the subcellular localization of the Na(+), K(+)-ATPase and its regulation by aldosterone depend on subunit isoform-specific structures, we expressed the cardiotonic steroid-sensitive human alpha isoforms 1-3 by retroviral transduction in mouse collecting duct mpkCCD(c14) cells. Each of the three exogenous human isoforms could be detected by Western blotting. Immunofluorescence indicated that the exogenous alpha1 subunit to a large extent localizes to the basolateral membrane or close to it, whereas much of the alpha2 subunit remains intracellular. An ouabain-sensitive current carried by exogenous pumps could be detected in apically amphotericin B-permeabilized epithelia expressing human alpha1 and alpha2 subunits, but not the alpha3 subunit. This current displayed a higher apparent Na(+) affinity in pumps containing human alpha2 subunits (10 mM) than in pumps containing human alpha1 (33.2 mM) or endogenous (cardiotonic steroid-resistant) mouse alpha1 subunits (mean: 16.3 mM). A very low mRNA level of the Na(+), K(+)-ATPase gamma subunit (FXYD2) in mpkCCD(c14) cells suggested that this ancillary gene product is not responsible for the relatively low apparent Na(+) affinity measured for a1 subunit-containing pumps. Aldosterone increased the pump current carried by endogenous pumps and by pumps containing the human alpha1 subunit. In contrast, the current carried by pumps with a human alpha2 subunit was not stimulated by the same treatment. In summary, quantitative basolateral localization of the Na(+), K(+)-ATPase and its responsiveness to aldosterone require alpha1 subunit-specific sequences that differentiate this isoform from the alpha2 and alpha3 subunit isoforms.