Malaoxon, the first and main metabolite of malathion, unlike its parent compound, can damage DNA in human lymphocytes, by mechanism(s) including oxidative action. Vitamin C may inhibit the DNA-damaging effect of malaoxon. To explore this effect further, we investigated the action of malaoxon on DNA in lymphocytes pretreated with a potent antioxidant, α-tocopherol (vitamin E), using the single-cell gel electrophoresis (comet assay). Addition of α-tocopherol to a final concentration of 20 and 80 μM reduced a dose-dependent DNA damaging effect of a 1-h incubation of human peripheral blood lymphocytes at 37°C with malaoxon at 25, 75, or 200 μM, measured as the increase in the comet tail moment of the lymphocytes. Lymphocytes treated with α-tocopherol displayed faster kinetics of removing damage to their DNA, and α-tocopherol abolished the cytotoxic effect of malaoxon at 200 μM. The protective action of α-tocopherol followed from its interaction with DNA or with malaoxon bound to DNA or lymphocytes rather than from chemical inactivation of malaoxon by it. Catalase, an enzyme inactivating hydrogen peroxide, decreased the extent of DNA damage induced by malaoxon. Lymphocytes exposed to malaoxon and treated with formamidopyrimidine-DNA glycosylase and 3-methyladenine-DNA glycosylase II, enzymes recognizing oxidized and methylated bases, displayed a greater extent of DNA damage than those not treated with these enzymes. The results suggest that hydrogen peroxide and reactive oxygen species may be involved in the formation of DNA lesions induced by malaoxon. Malaoxon can also methylate DNA bases, and α-tocopherol can be considered a protective agent against DNA damage in persons occupationally exposed to malathion/malaoxon.