The Na,K-ATPase undergoes conformational transitions during its catalytic cycle that mediate energy transduction between the phosphorylation and cation-binding sites. Structure-function studies have shown that transmembrane segments H5 and H6 in the alpha subunit of the enzyme participate in cation binding and transport. The Ca-ATPase crystal structure indicates that the H5 helix extends into the cytoplasmic ATP binding domain, finishing 4-5 A from the phosphorylation site. Here, we test whether the phosphorylation of the Na,K-ATPase leads to conformational changes in the cation-binding H5-H6 hairpin. Using as background an enzyme where all wild-type Cys in the transmembrane region were replaced, Cys were introduced in the joining loop and extracellular ends of H5 and H6. Mutated proteins were expressed in COS cells and probed with Hg(2+), [2-(trimethylammonium)ethyl]methanethiosulfonate (MTSET), and biotin-maleimide, applied to the extracellular media while placing the cells in two different media (K-medium and Na-medium). We assumed that under these treatment conditions most of the enzyme would be in one of two predominant conformations: E1 (K-medium) and E2P (Na-medium). The extent of enzyme inactivation by Hg(2+) or MTSET treatment was dependent on the targeted position; i.e., proteins carrying Cys in the outermost positions were more affected by treatment. Moreover, in the case of proteins carrying Cys at positions 785, 787, and 797, driving the enzyme to phosphorylated conformations (Na-media) led to a larger inactivation. Similarly, biotinylation of introduced Cys was also influenced by the enzyme conformation, with a larger extent of modification after treatment of cells in the Na-medium (E2P form). These results can be explained by the enzyme phosphorylation driving the outward movement of the H5 helix. Thus, they provide experimental evidence for a structure-function mechanism where, via H5, enzyme phosphorylation leads to a conformational change at the cation-binding site and the consequent cation translocation.