A soluble protein from Saccharomyces cerevisiae specifically provides protection against a thiol-containing oxidation system but not against an oxidation system without thiol. This 25 kDa protein was thus named thiol-dependent protector protein (TPP). The antioxidant role of TPP in the cellular defense against oxidative stress was investigated in a wild-type and a mutant yeast strain in which the tpp gene was disrupted by homologous recombination. Upon exposure in aerobic conditions to menadione, a well-known redox-cycling agent, there was a distinct difference between these two strains with regard to growth kinetics, viability, and superoxide dismutase activity. However, modulation of activities of other antioxidant enzymes, such as catalase and glutathione reductase, remained unaltered in both strains either with or without menadione. When the oxidation of 2',7'-dichlorofluorescin was used to examine hydroperoxide production in yeast cells, the tpp-null mutant showed a 2- to 3-fold increase in fluorescence upon exposure to menadione as compared to wild-type cells. The extent of damage to DNA, estimated by the increase of strand breaks and 8-hydroxy-2'-deoxyguanosine levels, in tpp mutant cells upon exposure to menadione was significantly higher than that of wild-type cells. These results suggest that TPP may play a direct role in cellular defense against oxidative stress by functioning as an antioxidant protein.