The focus of this article is on progress in establishing structure-function relationships through site-directed mutagenesis and direct binding assay of Tl⁺, Rb⁺, K⁺, Na⁺, Mg²⁺ or free ATP at equilibrium in Na,K-ATPase. Direct binding may identify residues coordinating cations in the E₂[2K] or E₁P[3Na] forms of the ping-pong reaction sequence and allow estimates of their contributions to the change of Gibbs free energy of binding. This is required to understand the molecular basis for the pronounced Na/K selectivity at the cytoplasmic and extracellular surfaces. Intramembrane Glu³²⁷ in transmembrane segment M4, Glu⁷⁷⁹ in M5, Asp⁸⁰⁴ and Asp⁸⁰⁸ in M6 are essential for tight binding of K⁺ and Na⁺. Asn³²⁴ and Glu³²⁷ in M4, Thr⁷⁷⁴, Asn⁷⁷⁶, and Glu⁷⁷⁹ in 771-YTLTSNIPEITP of M5 contribute to Na⁺/K⁺ selectivity. Free ATP binding identifies Arg⁵⁴⁴ as essential for high affinity binding of ATP or ADP. In the 708-TGDGVND segment, mutations of Asp⁷¹⁰ or Asn⁷¹³ do not interfere with free ATP binding. Asp⁷¹⁰ is essential and Asn⁷¹³ is important for coordination of Mg²⁺ in the E₁P[3Na] complex, but they do not contribute to Mg²⁺ binding in the E₂P-ouabain complex. Transition to the E₂P form involves a shift of Mg²⁺ coordination away from Asp⁷¹⁰ and Asn⁷¹³ and the two residues become more important for hydrolysis of the acyl phosphate bond at Asp³⁶⁹.