In order to clarify the relationship between the salt tolerance of Zygosaccharomyces rouxii and the function of Na+-ATPase, a gene which exhibited homology to the Na+-ATPase gene (ENA1) of Saccharomyces cerevisiae was isolated from Z. rouxii. This newly isolated gene (ZENA1) encoded a product of 1048 amino acids. The predicted amino-acid sequence of Zena1p was highly homologous to that of S. ceravisiae Ena1p and Ena2p, and Schwanniomyces occidentalis Ena1p and Ena2p, but showed low homology to that of Zpma1p, which is the product of the Z. rouxii plasma membrane H+-ATPase gene (ZPMA1). Zena1p shares the peptide motifs which have been suggested to participate in the function of ATPase. Expression of ZENA1 was observed, but was independent of NaCl shock. When ZENA1 was expressed in salt-sensitive S, cerevisiae under the regulation of a GAL1 promoter by using the expression vector pYES2, salt tolerance of the transformants was observed, The growth characteristics of Zena1 Delta-disruptants of Z. rouxii and the pH profiles of their plasma membrane ATPase activity were almost the same as those of the wild-type strain, indicating that the function of Zena1p is of little relevance to the salt tolerance property of Z. rouxii, By considering the close relationship between the salt tolerance of Z. rouxii and the function of its Na+/H+-antiporter, we concluded that the extrusion of Nat across the plasma membrane in Z. rouxii cells might be carried out mainly via the function of the Na+/H+-antiporter In a high salinity environment.