Several ethanol-tolerant mutants have been bred from industrial sake yeasts, but the mechanism of ethanol tolerance in these mutants has not been elucidated. After the determination of the entire genome sequence of Saccharomyces cerevisiae, various methods to monitor the whole-gene expression of the yeast have been developed. In this study, we used a commercially available nylon membrane on which virtually every gene of S. cerevisiae was spotted to compare expression profiles between the ethanol-tolerant mutant and its parent sake yeast to investigate the mechanism of ethanol tolerance in this mutant. As a result, we found that several genes were highly expressed only in the ethanol-tolerant mutant but not in the parent strain. These genes were known to be induced in cells that were exposed to various stresses, such as ethanol, heat, and high osmolarity, or at the stationary-phase but not at the log-phase. In the ethanol-tolerant mutant, the expression level of these stress-responsive genes was further increased after exposure to ethanol. We also found that substances such as catalase, glycerol and trehalose that may have protective roles under stressful conditions were accumulated in high amounts in the ethanol-tolerant mutant. The ethanol-tolerant mutant also exhibited resistance to other stresses including heat, high osmolarity and oxidative stress in addition to ethanol tolerance. These results indicate that the mutant exhibits multiple stress tolerance because of elevated expression of stress-responsive genes, resulting in accumulation of stress protective substances.