This study is to investigate the ethanol tolerance of yeast strains related to fatty acid composition and intracellular ethanol concentration for various fermentation temperatures. The maximum accumulation of ethanol in the cells was decreased by lowering the fermentation temperature, while unsaturated fatty acid content was increased by decreasing the fermentation temperature. Thus, we found that the increase of ethanol accumulation in cells resulted in the decrease of unsaturated fatty acid content. Therefore, it was suggested that the composition of unsaturated fatty acids in the cell membrane be strongly related to the diffusion of ethanol from cell to medium.