This study deals with investigation of the ethanol tolerance of yeast strains with respect to fatty acid composition and intracelluar ethanol concentration during alcohol fermentation. The cell viabilities and fermentation abilities of Saccharomyces cerevisiae and Kluyveromyces fragilis were improved by aeration and addition of unsaturated fatty acids into growth medium. Aeration decreases the accumulation of ethanol, while increases unsaturated fatty acid contents inside yeast cells. Thus it was found that oxygen and unsaturated fatty acids play decisive roles in the increase of ethanol tolerance of yeasts.