The Na,K-ATPase is specifically inhibited by the cardiac glycoside, ouabain. Via a largely undefined mechanism, the ouabain affinity of the Na,K- ATPase can be manipulated by mutating the residues at the borders of the first extracellular (M1-M2) loop of the α subunit [Price, E. M., Rice, D. A., and Lingrel, J. B. (1990) J. Biol. Chem. 265, 6638-6641]. To address this issue, we compared the effects of two combinations of charged residues at the M1-M2 loop border, R113,D124 and D113,R124 (numbered according to the rat α1 subunit), on the ouabain sensitivity of the α1 and α2 isoforms. We report that ouabain sensitivity is dependent not only upon the identity of the residues at the M1-M2 loop border but also upon the context into which they are introduced. Furthermore, at low concentrations of ATP, the identity of the residues at the M1-M2 loop border affects the regulation of ATP hydrolysis by potassium in an isoform-specific manner. Analysis of chimeric α subunits reveals that the effects of potassium are determined primarily by the interaction of the N-terminus and M1-M2 loop with the C-terminal third of the α subunit. M1-M2 loop border residues may, therefore, influence ouabain sensitivity indirectly by altering the stability or structure of the intermediate of the Na,K-ATPase catalytic cycle which is competent to bind ouabain.