The intramembrane GLU781 residue of the Na,K-ATPase a subunit has been postulated to have a role in the binding and/or occlusion of cations. To ascertain the role of Glu781, the residue was substituted with an aspartate, alanine, or lysine residue and the mutant Na,K-ATPases were coexpressed with the native β1 subunit in Sf9 insect cells using the baculovirus expression system. All α mutants are able to efficiently assemble with the β1 subunit and produce catalytically competent Na,K- ATPase molecules with hydrolytic activities comparable to that of the wild- type enzyme. Analysis of the kinetic properties of the mutated enzymes showed a decrease in apparent affinity for K+ compared to wild-type Na,K-ATPase, with the lysine and alanine substitutions displaying the greatest reduction. All Na,K-ATPase mutants demonstrated a significant increase in apparent affinity for ATP compared to wild.type Na,K-ATPase, while the sensitivity to the cardiotonic inhibitor, ouabain, was unchanged. The dependence on Na+, however, differs among the mutant enzymes with both the Glu781 → Asp and Glu781 → Ala mutants displaying a decrease in the apparent affinity for the cation, while the Glu781 → Lys mutant exhibits a modest increase. Furthermore, in the absence of K+, the Glu781 → Ala mutant displays a Na+-ATPase activity and a cellular Na+ influx suggesting that Na+ is substituting for K+ at the extracellular binding sites. The observation that trypsin digestion of the Glu781 → Ala mutant in Na+ medium produces a K+- stabilized tryptic fragment also intimates a decreased capacity of the mutant to discriminate between Na+ and K+ at the extracellular loading sites. All together, these data implicate Glu781 of the Na,K-ATPase a subunit as an important coordinate of cation selectivity and activation, although the modest effect of Glu781 → Lys substitution seemingly precludes direct involvement of the residue in the cation binding process. In addition, the fifth membrane segment is proposed to represent an important communicative link between the extramembraneous ATP binding domain and the cation transport regions of the Na,K-ATPase.