Background: Antioxidant supplementation with vitamin E had no effect in the prevention of cardiovascular diseases (CVD) in three recent large, randomized clinical trials. In order to reassess critically the role of vitamin E in CVD prevention, it is important to establish whether these results are related to a lack of antioxidant action. Methods: We examined the in vivo antioxidant effect of vitamin E (300 mg/day for about three years) in 144 participants in the Primary Prevention Project (females and males, aged greater than or equal to 50 y, with at least one major CV risk factor, but no history of CVD). Urinary 8-epi-PGF(2alpha) (isoprostane F-2alpha-III or 15-F-2t-isoP), a validated biomarker of lipid peroxidation, was measured by mass spectrometry. Results: Urinary excretion of 8-epi-PGF(2alpha) [pg/mg creatinine, median ( range)] was 141 (67-498) in treated and 148 (76-561) in untreated subjects (p = 0.10). Taking into account possible confounding variables, multiple regression analysis confirmed that vitamin E had no significant effect on this biomarker. Levels of 8-epi-PGF(2alpha) were in the normal range for most subjects, except smokers and those with uncontrolled blood pressure or hyperglycemia. Conclusions: Prolonged vitamin E supplementation did not reduce lipid peroxidation in subjects with major cardiovascular risk factors. The observation that the rate of lipid peroxidation was near normal in a large proportion of subjects may help explain why vitamin E was not effective as an antioxidant in the PPP study and was ineffective for CVD prevention in large scale trials.