Mutations in the mitochondrial genome leading to mitochondrial dysfunction have been reported in a variety of cancers. However, the potential implication of these findings in the cellular response to cancer therapeutic agents is unclear. To examine the importance of mitochondrial DNA (mitDNA) encoded functions in cancer therapeutic response, we determined the clonogenic survival of HSL2 (Rho+, HeLa subline), and its derivative cell line lacking mitDNA (Rho0) after exposure to different anticancer agents. We found that isogenic Rho0 cells lacking mitDNA were extremely resistant to adriamycin and photodynamic therapy (PDT) induced cell death, whereas the Rho+ cell line was sensitive. However, there was no measurable difference in the responses of these cell lines to either alkylating agent or gamma-radiation. We show that the development of resistance to adriamycin was not due to changes in apoptotic cell death, cell cycle response or to the uptake of adriamycin in isogenic Rho0 cells. We also demonstrate that exposure of HeLa cells to adriamycin leads to mutations in mitDNA. These studies provide direct evidence that mitDNA plays an important role in cellular sensitivity to cancer therapeutic agents.