The frequency of ethanol-induced respiratory deficient mutants and lipid composition in two Saccharomyces cerevisiae strains showing different degrees of ethanol tolerance were investigated. The more ethanol-tolerant strain exhibited a lower frequency of ethanol-induced respiratory deficient mutants than the less ethanol-tolerant strain. In addition, the more ethanol-tolerant strain contained a higher ergosterol/phospholipid ratio, a higher proportion of phosphatidylcholine, a lower proportion of phosphatidylethanolamine, a higher incorporation of long-chain fatty acids in total phospholipids, and a slightly higher proportion of unsaturated fatty acids in total phospholipids than the less ethanol-tolerant strain. These results show a clear relationship between the lipid composition, the frequency of ethanol-induced respiratory deficient mutants, and the ethanol tolerance of S. cerevisiae. A possible explanation of this relationship is discussed.