Fully active Na,K-ATPase and lethal mutations can be expressed in yeast cells in yields allowing for equilibrium ATP binding, occlusion of T1⁺, K⁺ displacement of ATP, and Na⁺-dependent phosphorylation with determinations of affinity constants for binding and constants for the conformational equilibria. Removal of the charge and hydrophobic substitution of the phosphorylated residue (Asp³⁶⁹Ala) reveals an intrinsic high affinity for ATP binding (K(d) 2.8 vs. 100 nM for wild type) and causes a shift of conformational equilibrium towards the E₂ form. Substitution of Glu³²⁷, Glu⁷⁷9 Asp⁸⁰⁴ or Asp⁸⁰⁸ in transmembrane segments 4, 5, and 6 shows that each of these residues are essential for high-affinity occlusion of K⁺ and for binding of Na⁺. Substitution of other residues in segment 5 shows that the carboxamide group of Asn⁷⁷⁶ is important for binding of both K⁺ and Na⁺. Differential effects of the relevant mutations identify Thr⁷⁷⁴ as specific determinant of Na⁺ binding in the E₁P[3Na] form, whereas Ser⁷⁷⁵ is a specific participant of high-affinity binding of the E₂[2K] form, suggesting that these residues engage in formation of a molecular Na⁺/K⁺ switch. The position of the switch may be controlled by rotating or tilting the helix during the E₁-E₂ transition.