The ion-transporting H,K-ATPase and Na,K-ATPase enzymes are each composed of an alpha and a beta subunit. It is known that assembly of the alpha and beta subunits of the Na,K-ATPase is necessary for the cell-surface delivery of the active enzyme. We have examined the molecular domains involved in the assembly of the H,K-ATPase and Na,K-ATPase alpha and beta subunits by expressing individual subunits and subunit chimeras in transiently transfected COS-1 cells. Our results demonstrate that the H,K-ATPase alpha subunit requires its beta subunit for efficient cell-surface expression, as determined by indirect immunofluorescence. The H,K-ATPase beta protein appears to be able to get to the cell surface unaccompanied by any alpha subunit and appears to localize as well to a population of intracellular vesicles. We find that a transfected chimera encoding the NH2-terminal half of the H,K-ATPase alpha subunit and the COOH-terminal half of the Na,K-ATPase alpha subunit appears to assemble with the endogenous Na,K-ATPase beta subunit and to reach the plasmalemma. Transfection of the complementary alpha chimera requires coexpression with the H,K-ATPase beta subunit in order to attain surface delivery. Thus, it is the COOH-terminal half of the alpha subunit that specifies assembly with a particular beta subunit.