In order to investigate the role of mitochondrial DNA (mtDNA) in human breast cancer cell proliferation and apoptosis, a mtDNA-deficient cell line, T47D rho(0), was generated following a long-term exposure to ethidium bromide (EtBr). T47D rho(0) cells showed a marked decrease in mitochondrial membrane potential (DeltaPsi(m)). However, the apoptosis rate of T47D rho(0) cells was the same as that of their parental cells, suggesting that the change in DeltaPsi(m) was insufficient to induce cell death. Electromicroscopy revealed a profound alteration of mitochondrial morphology, which was consistent with the loss of mtDNA and the decrease in DeltaPsi(m). Disruption of mtDNA resulted in a slower proliferation rate in tissue culture and a reduction in anchorage-independent growth. An in vivo assay revealed a severe impairment of tumorigenicity in T47D rho(0) cells, indicating the biological relevance of in vitro studies. Taken together, our results suggest that the integrity of mtDNA plays a critical role in human breast cancer cell proliferation and tumorigenesis.