Aims: To study the effect of prestress conditions on the freezing and thawing (FT) response of two baker's yeast strains and the use of statistical analysis to optimize resistance to freezing. Methods and Results: Tolerance to FT of industrial strains of Saccharomyces cerevisiae was associated to their osmosensitivity and growth phase. Pretreatments with sublethal stresses [40 degrees C, 0.5 mol l(-1) NaCl, 1.0 mol l(-1) sorbitol or 5% (v/v) ethanol] increased freeze tolerance. Temperature or hyperosmotic prestresses increased trehalose contents, nevertheless no clear correlation was found with improved FT tolerance. Plackett-Burman design and response surface methodology were applied to improve freeze tolerance of the more osmotolerant strain. Optimal prestress conditions found were: 0.779 mol l(-1) NaCl, 0.693% (v/v) ethanol and 32.15 degrees C. Conclusions: Ethanol, saline, osmotic or heat prestresses increased freezing tolerance of two phenotypically distinct baker's yeast strains. A relationship among prestresses, survival and trehalose content was not clear. It was possible to statistically find optimal combined prestress conditions to increase FT tolerance of the osmotolerant strain. Significance and Impact of the Study: Statistically designed combination of prestress conditions that can be applied during the production of baker's yeast could represent a useful tool to increase baker's yeast FT resistance.