Intrinsic oxidative stress through increased production of reactive oxygen species (ROS) is associated with carcinogenic transformation, cell toxicity, and DNA damage. Mitochondrial DNA (mtDNA) is a natural surrogate to oxidative DNA damage. MtDNA damage results in the loss of its supercoiled structure and is readily detectable using a novel, supercoiling-sensitive real-time PCR method. Our studies have demonstrated that mtDNA damage, as measured by DNA strand breaks and copy number depletion, is very sensitive to exogenous H2O2 but independent of endogenous ROS production in both prostate cancer and normal cells. In contrast, aggressive prostate cancer cells exhibit a more than 10-fold sensitivity to H2O2-induced cell toxicity than normal cells, and a cascade of secondary ROS production is a critical determinant to the differential response. We propose a new paradigm to account for different mechanisms governing cellular oxidative stress, cell toxicity, and DNA damage with important ramifications in devising new techniques and strategies in prostate cancer prevention and treatment.