As a basis for an evaluation of the role of the cellular antioxidant defense system against oxidative stress, the effects of an organic hydroperoxide, tertiary-butyl hydroperoxide (t-BuOOH), on the activity of antioxidant enzymes were investigated in cultured Chinese hamster V79 cells. Incubation of cells with t-BuOOH for 1 h significantly increased the activity of Cu-Zn superoxide dismutase (SOD) up to a level 1.4 times that of control cells. In contrast, the activities of catalase and glutathione reductase (GSSG-Rx) were not affected, while the activity of glutathione peroxidase (GSH-Px) was inhibited to a significant extent by t-BuOOH. Hydrogen peroxide also inhibited GSH-Px activity but its potency in this regard was somewhat lower than that of equimolar amount of t-BuOOH. Earlier studies demonstrated that t-BuOOH-induced cytotoxicity, single strand breaks (ssb) in DNA and structural aberrations in the chromosomes of V79 cells can be suppressed almost completely by an iron chelator o-phenanthroline. However, the iron chelator did not suppress the t-BuOOH-induced inhibition of GSH-Px activity. Likewise, a diffusible scavenger of free radicals, butylated hydroxytoluene (BHT) did not affect the hydroperoxide-induced inhibition of the enzymatic activity. These results suggest that a mechanism other than iron-mediated radical reaction is involved in the inhibition of GSH-Px activity by t-BuOOH. Modulation of the activity of antioxidant enzymes by the oxidative agent diamide was very similar to that by t-BuOOH. Inhibition of GSH-Px activity by t-BuOOH was reversible and the reduced activity returned to pre-inhibition levels within 1-2 h of post-treatment incubation. A mechanism for the inhibition of GSH-Px by t-BuOOH is discussed with reference to the oxidation of selenocysteine residues which results in perturbation of the normal catalytic cycle.