Oxidative DNA damage may be important in mutagenic, carcinogenic, and aging processes. Although it is plausible that antioxidant vitamins may reduce oxidative DNA damage, evidence from human studies has been sparse and inconsistent. We determined the short-term effects of vitamin C (500 mg/day) and vitamin E (400 IU d-alpha-tocopheryl acetate/day) supplements on oxidative DNA damage in a double-masked, placebo-controlled, 2x2 factorial trial in 184 nonsmoking adults. Mean duration of supplementation was 2 months. Oxidative DNA damage was measured by 24-h urinary excretion of 8-hydroxy-2'-deoxyguanosine (8-OHdG). At baseline, urinary 8-OHdG (mean +/- SE; ng/mg creatinine) was associated with race (15.6 +/- 0.8 in African Americans versus 20.3 +/- 1.2 in Caucasians, P = 0.001), prior antioxidant supplement use (18.6 +/- 0.8 in users versus 13.8 +/- 1.5 in non-users, P = 0.007), and regular exercise (19.2 +/- 1.1 in exercisers versus 16.6 +/- 0.9 in non-exercisers, P = 0.04). Fruit and vegetable intake and serum ascorbic acid were inversely associated with urinary 8-OHdG (P-trend = 0.02 and 0.016, respectively). The benefits of fruit and vegetable intake became evident with the consumption being at least three servings/day. At the end of supplementation, change from baseline in urinary 8-OHdG (mean +/- SE; ng/mg creatinine) was -0.6 +/- 1.4 (P = 0.61), 0.6 +/- 1.1 (P = 0.59), 0.5 +/- 1.0 (P = 0.61), and 1.6 +/- 1.4 (P = 0.27) in the placebo, vitamin C alone, vitamin E alone, and combined vitamins C and E groups, respectively. In overall and subgroup analyses, there was no significant main effect or interaction effect of the supplements on urinary 8-OHdG. In conclusion, supplementation of diet with vitamin C (500 mg/day) and vitamin E (400 IU d-alpha-tocopheryl acetate/day) had no significant main effect or interaction effect on oxidative DNA damage as measured by urinary 8-OHdG in nonsmoking adults. However, several aspects of a healthy lifestyle were associated with lower oxidative DNA damage.