Vitamin E is the major lipid-soluble chain-breaking antioxidant in mammals and plays an important role in normal development and physiology. Deficiency (whether dietary or genetic) results in primarily nervous system pathology, including cerebellar neurodegeneration and progressive ataxia (abnormal gait). However, despite the widely acknowledged antioxidant properties of vitamin E, only a few studies have directly correlated levels of reactive oxygen species with vitamin E availability in animal models. We explored the relationship between vitamin E and reactive oxygen species in two mouse models of vitamin E deficiency: dietary deficiency and a genetic model (tocopherol transfer protein, Ttp-/- mice). Both groups of mice developed nearly complete depletion of α-tocopherol (the major tocopherol in vitamin E) in most organs, but not in the brain, which was relatively resistant to loss of α-tocopherol. F4-neuroprostanes, an index of lipid peroxidation, were unexpectedly lower in brains of deficient mice compared with controls. In vivo oxidation of dihydroethidium by superoxide radical was also significantly lower in brains of deficient animals. Superoxide production by brain mitochondria isolated from vitamin E-deficient and Ttp-/- mice, measured by electron paramagnetic resonance spectroscopy, demonstrated a biphasic dependence on exogenously added α-tocopherol. At low concentrations, α-tocopherol enhanced superoxide flux from mitochondria, a response that was reversed at higher concentrations. Here we propose a mechanism, supported by molecular modeling, to explain decreased superoxide production during α-tocopherol deficiency and speculate that this could be a beneficial response under conditions of α-tocopherol deficiency.