Trehalose is an enigmatic compound that accumulates in Saccharomyces cerevisiae and has been implicated in survival under various stress conditions by acting as membrane protectant, as a supplementary compatible solute or as a reserve carbohydrate that may be mobilized during stress. In this study, specific mutants in trehalose metabolism were used to evaluate whether trehalose contributes to survival under severe osmotic stress and generates the compatible solute glycerol under moderate osmotic stress. The survival under severe osmotic stress (0.866 a(w), NaCl or sorbitol) of mutants was compared to that of the wild-type strain when cultivated to either the mid, exponential or the stationary growth phase on glucose, galactose or ethanol. Stationary-phase cells survived better than exponential-phase cells. The death rates of ethanol-grown cells were lower than those of galactose-grown cells, which in turn survived better than glucose-grown cells. There was a strong relationship between intracellular trehalose levels and resistance to osmotic stress. The mutant strains unable to produce trehalose (tps1 Delta tps2 Delta and tps1 Delta hxk2 Delta) were more sensitive to severe osmotic stress (0.866 a(w)) than the isogenic wild-type strain, confirming a role for trehalose in survival. Hyperaccumulation of trehalose found in the nth1 Delta and the nth1 Delta gpd1 Delta mutant strains, however, did not improve survival rates compared to the wildtype strain. When wild-type, nth1 Delta and nth1 Delta gpd1 Delta cells were exposed to moderate osmotic stress (0.98 and 0.97 a(w), NaCl), which permits growth, glycerol production did not appear to be related to the intracellular trehalose levels although glycerol levels increased more rapidly in nth1 Delta cells than in wild-type cells during the initial response to osmotic stress. These data indicate that trehalose does not act as a reserve compound for glycerol synthesis under these conditions. No evidence was found for solutes other than glycerol and trehalose being significant for the survival of or growth by S. cerevisiae under osmotic stress conditions.