Intracellular glycerol content affects the freeze-thaw stress tolerance of Saccharomyces cerevisiae. We have recently reported that intracellular-glycerol-enriched cells cultured in glycerol medium acquire tolerance to freeze stress and retain high leavening ability even in dough after frozen storage [Izawa et al. ( 2004) Appl Microbiol Biotechnol http:// dx. doi. org/ 10.1007/ s00253-004- 1624-4]. A deletion mutant of the FPS1 gene, which encodes a glycerol channel, accumulates glycerol inside the cell without an exogenous supply of glycerol into the medium. We found that the fps1Delta cells acquired tolerance to freeze stress and retained high leavening ability in dough after frozen storage for 7 days. These results suggest that the fps1Delta mutant is a useful strain for developing better frozen-dough with a commercial advantage.