This paper summarizes experiments concerned with the functional consequences of mutations in cytoplasmic regions of the alpha 1 subunit of the Na,K-ATPase, in particular the amino terminus, the first cytoplasmic loop between transmembrane segments M2 and M3, and the major cytoplasmic loop between M4 and M5. In the first mutation (alpha 1M32), 32 residues were removed from the N-terminus. The second mutation (E233K) was in the putative beta strand of M2-M3 loop and the third, comprised the replacement of the amino terminal half of loop M4-M5 of the Na,K-ATPase with the homologous segment (residues 356-519) of the gastric H,K-ATPase. The first two mutations, either separately or in combination (alpha 1M32E233K), shift the equilibrium between the major conformational states of the enzyme, E1 and E2, in favor of E1 as manifested by increased apparent affinity for ATP, lower catalytic turnover, and decreased sensitivity to inhibition by vanadate. The striking changes observed with alpha 1M32E233K suggests interactions between the N-terminus, the beta-strand in the M2-M3 loop and the catalytic phosphorylation site. The behavior of these mutants contrasts with that of least one mutant involving substitution of a residue in the putative cation binding pocket, namely S775A in the fifth transmembrane segment (Arguello, J.M., & Lingrel, J. B. J. Biol. Chem. 270: 22764-22771, 1995). Although its K+/ATP antagonism resembles that of the foregoing cytoplasmic mutants, its vanadate sensitivity is unaltered suggesting that changes in apparent affinity for ATP are secondary to changes in K+ ligation. The question of cation selectivity, in particular that of Na+ versus protons, has been addressed in structure/function analysis of a cytoplasmic chimera involving the M4-M5 loop. Transport studies performed in the presence or absence of Na+ and at low versus high pH indicate a marked alteration in cation affinity and/or selectivity. This results suggests coupling of an alteration in the large M4-M5 cytoplasmic domain to cation binding in, presumably, the juxtapositioned transmembrane domain.