We investigated the effect of long-term vitamin E deficiency (38 weeks) on free radical (superoxide) production and free radical products (neuroprostanes and isoprostanes) and on mitochondrial function (oxygraph and electron transport chain activities) in C57B6J mice. We found that after 38 weeks, while liver was approximately 95% deficient, the brain had retained approximately 50% of its alpha-tocopherol. We also found that superoxide production was lowered in multiple brain regions of male vitamin E-deficient mice, as were neuroprostanes. Oxygraph studies showed higher respiratory control ratios (RCRs) in liver and lower RCRs in brain, which did not appear to be due to changes in electron transport chain activities. We conclude that vitamin E can function in vivo in both its traditional role as a lipid-soluble antioxidant as well as in non-traditional roles in the mitochondria.