Studies of structure-function relationships in Na,K-ATPase require high yield expression of inactive mutations in cells without endogenous Na,K- ATPase activity. In this work we developed a host/vector system for expression of fully active pig Na,K-ATPase as well as the inactive mutations D369N and D807N at high levels in Saccharomyces cerevisiae. The α1- and β1- subunit cDNAs were inserted into a single 2-μm-based plasmid with a high and regulatable copy number and strong galactose-inducible promoters allowing for stoichiometric alterations of gene dosage. The protease-deficient host strain was engineered to express high levels of GAL4 transactivating protein, thereby causing a 10-fold increase in expression to 32,500 ± 3,000 [³H]ouabain sites/cell. In one bioreactor run 150-200 g of yeast were produced with 54 ± 5 μg of Na,K-pump protein/g of cells. Through purification in membrane bound form the activity of the recombinant Na,K- ATPase was increased to 42-50 pmol/mg of protein. The Na,K dependence of ATP hydrolysis and the molar activity (4,500-7,000 min^-1) were close to those of native pig kidney Na,K-ATPase. Mutations to the phosphorylation site (D369N) or presumptive cation sites (D807N), both devoid of Na,K-ATPase activity, were expressed in the yeast membrane at the same α-subunit concentration and [³H]ouabain binding capacity as the wild type Na,K- ATPase. The high yield and absence of endogenous activity allowed assay of [³H]ATP binding at equilibrium, demonstrating a remarkable 18-fold increase in affinity for ATP in consequence of reducing the negative charge at the phosphorylation site (D369N).