The alcohol fermentation was carried out to study the effect of aeration and unsaturated fatty acids added on the ethanol tolerance of Saccharomyces cerevisiae STV89 and Kluyveromyces fragilis CBS397. The cell growth rate and ethanol production rate was stimulated by aeration and the cell mass production and ethanol production were also substantially improved. With respect to strains, the maximum specific growth rate and overall ethanol productivity of K. fragilis under aerated condition were 6.4 fold and 4.4 fold higher than those of strictly anaerobic condition, although those of S. cerevisiae were increased 1.7 times and 2.3 times by aeration. The addition of ergosterol, linoleic acid and oleic acid also improved the cell growth and ethanol production of S. cerevisiae and K. fragilis. Thus it was found that oxygen and unsaturated fatty acids added played a decisive role on the increase of ethanol tolerance of yeast strains.