Zygosaccharomyces rouxii mutants defective in NaCl tolerance were isolated. One of the mutants, 77-1S, could not grow in a medium containing 1% (w/v) NaCl at pH 6.0, but showed recovery of 15% NaCl tolerance at pH 4.0 in the same medium. The mutant 77-1S leaked intracellular glycerol through the plasma membrane (PM) under 10% NaCl at pH 6.0, but the glycerol leakage decreased under the 10% NaCl-tolerant condition at pH 4.0. The intracellular content of Na+ in 77-1S cells grown in YPD medium containing 0.8% NaCl at pH 6.0 was about ninefold higher than that of the parent M7, whereas the Na+ content was similar in the mutant and the parent at pH 4.0. The leakage of glycerol and the influx of Na+ through the PM under the NaCl condition were considered to be caused by damage to the PM in the mutant 77-1S. Neither an increase of oleic acid nor a decrease of linoleic acid was observed under the 10% NaCl condition at pH 6.0 in the mutant 77-1S, although such changes were seen in the parent M7. The activity of PM H+-ATPase prepared from 77-1S grown at pH 6.0 was almost the same as that of the parent M7. The mutant 77-1S showed both an increase of oleic acid and a decrease of linoleic acid in the presence of 10% NaCl at pH 4.0 in the conditions under which the strain could grow. Therefore, lipid saturation seems to be important for salt tolerance in Z. rouxii cells under a high-NaCl condition.