The role of trehalose as cell protector against oxidative stress induced by H(2)O(2) has been studied in Saccharomyces cerevisiae mutants in which the two trehalase genes ATH1 and NTH1 are deleted. The addition of low H(2)O(2) concentrations to proliferating cultures of either strain did not harm cell viability and induced a marked activity to Nth1p, but with no significant level of trehalose accumulation. This pattern was reversed after a more severe H(2)O(2) treatment that caused drastic cell killing. The most severe phenotype corresponded to the Delta nth1 mutant. Under these conditions, the increase in Nth1p was abolished and a three-fold rise in trehalose content was recorded concomitant with activation of the trehalose synthase complex. The behavior of the double-disruptant Delta ath1Delta nth1 mutant was identical to that of wild-type cells, although in exponential cultures Ath1p activity was virtually undetectable upon exposure to H(2)O(2). Furthermore, these strains displayed an adaptive response to oxidative stress that was independent of intracellular trehalose synthesis. Our data strongly suggest that trehalose storage in budding yeasts is not an essential protectant in cell defense against oxidative challenge.