Short-term aldosterone coordinately regulates the cell-surface expression of luminal epithelial sodium channels (ENaC) and of basolateral Na+ pumps (Na,K-ATPase 1-1) in aldosterone-sensitive distal nephron (ASDN) cells. To address the question 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 isoforms 1-3 by retroviral transduction in mouse collecting duct mpkCCDc14 cells. Each of the three exogenous human isoforms could be detected by Western blotting. Immunofluorescence indicated that the exogenous 1 subunit to a large extent localizes to the basolateral membrane or close to it, whereas much of the 2 subunit remains intracellular. An ouabain-sensitive current carried by exogenous pumps could be detected in apically amphotericin B-permeabilized epithelia expressing human 1 and 2, but not 3 subunit. This current displayed a higher apparent Na+ affinity in the case of pumps containing human 2 (10 mM) than human 1 (33.2 mM) or endogenous (cardiotonic steroid-resistant) mouse 1 subunit (mean: 16.3 mM). A very low mRNA level of the Na,K-ATPase subunit (FXYD2) in mpkCCDc14 cells suggested that this ancillary gene product is not responsible for the relatively low apparent Na+ affinity measured for 1 subunit containing pumps. Aldosterone increased the pump current carried by endogenous pumps and by pumps containing the human 1 subunit. In contrast, the current carried by pumps with a human 2 subunit was not stimulated by the same treatment. In summary, quantitative basolateral localization of the Na,K-ATPase and its responsiveness to aldosterone require 1 subunit-specific sequences that differentiate this isoform from the 2 and 3 subunit isoforms.