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 + , Mg 2 + or free ATP at equilibrium in Na,K-ATPase. Direct binding may identify residues coordinating cations in the E 2 [2K] or E 1 P[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 Glu 3 2 7 in transmembrane segment M4, Glu 7 7 9 in M5, Asp 8 0 4 and Asp 8 0 8 in M6 are essential for tight binding of K + and Na + . Asn 3 2 4 and Glu 3 2 7 in M4, Thr 7 7 4 , Asn 7 7 6 , and Glu 7 7 9 in 771-YTLTSNIPEITP of M5 contribute to Na + /K + selectivity. Free ATP binding identifies Arg 5 4 4 as essential for high affinity binding of ATP or ADP. In the 708-TGDGVND segment, mutations of Asp 7 1 0 or Asn 7 1 3 do not interfere with free ATP binding. Asp 7 1 0 is essential and Asn 7 1 3 is important for coordination of Mg 2 + in the E 1 P[3Na] complex, but they do not contribute to Mg 2 + binding in the E 2 P-ouabain complex. Transition to the E 2 P form involves a shift of Mg 2 + coordination away from Asp 7 1 0 and Asn 7 1 3 and the two residues become more important for hydrolysis of the acyl phosphate bond at Asp 3 6 9 .