The ENA1 gene of Saccharomyces cerevisiae encodes a putative ATPase necessary for Na+ efflux. Plasma membranes and intracellular membranes of a yeast strain overexpressing the ENA1 gene contain significant amounts of ENA1 protein. Consequences of the overexpression with reference to the wild-type strain are: (1) a 5-fold higher content of the ENA1-protein in plasma membranes; (2) lower Na+ and Li+ effluxes; (3) slightly higher Na+ tolerance; and (4) much higher Li+ tolerance. The ENA1-specific ATPase activity in plasma membrane preparations of the overexpressing strain was low, but an ENA1 phosphoprotein was clearly detected when the plasma membranes were exposed to ATP in the presence of Na+ or to Pi in the absence of Na+. The characteristics of this phosphoprotein, which correspond to the acyl phosphate intermediaries of P-type ATPases, the absolute requirement of Na+ or other alkali cations for phosphorylation, and the Na+ and pH dependence of phosphorylation from ATP and Pi suggest that the product of the ENA1 gene may be a Na,H-ATPase, which can also pump other alkali cations. The role of the intracellular membranes structures produced with the overexpression of ENA1 in Na+ and Li+ tolerances and the existence of a beta-subunit of the ENA1 ATPase are discussed.