Depletion of the mitochondrial genome is involved in several human diseases, as well as in mitochondrial diseases induced by drug therapies used in the treatment of cancer and human immunodeficiency virus. In order to identify the molecular changes underlying the pathogenesis of mitochondrial diseases, we determined the oxidative status of a human cell line following depletion of the mitochondrial genome (denoted ρ0 cells). Our analysis revealed that ρ0 cells contained ∼10-fold lower levels of superoxide than parental cells (ρ+), as detected by oxidation of dihydroethidium. No concurrent decrease in oxidation of hydrogen peroxide, detected using the dye dichloroflorescein diacetate, was observed in ρ0 cells. Depletion of the mitochondrial genome did not affect either the expression of superoxide dismutase or its activity. However, catalase expression and its activity decreased in ρ0 cells. In addition, glutathione peroxidase activity was higher in ρ0 cells compared with ρ+. ρ0 cells showed increased lipid peroxidation, increased oxidative damage to the nuclear genome and impaired DNA repair. Our data illustrate the importance of the mitochondrial genome and its function to the cellular oxidative environment and nuclear genome instability. It also provides insights into the development of mitochondrial disease as a consequence of cancer therapy.