Mitochondrial DNA (mtDNA) is exposed to reactive oxygen species (ROS) produced during oxidative phosphorylation. Accumulation of several kinds of oxidative lesions, including oxidized pyrimidines, in mtDNA may lead to structural genomic alterations, mitochondrial dysfunction and associated degenerative diseases. In Escherichia coli, oxidative pyrimidines are repaired by endonuclease III (EndoIII) and endonuclease VIII (EndoVIII). To determine whether the overexpression of two bacterial glycosylase/AP lyases which predominantly remove oxidized pyrimidines from DNA, could improve mtDNA repair and cell survival, we constructed vectors containing sequences for the EndoIII and EndoVIII downstream of the mitochondrial targeting sequence (MTS) from manganese superoxide dismutase (MnSOD) and placed them under the control of the tetracycline (Tet)-response element. Successful integrations of MTS-EndoIII or MTS-EndoVIII into the HeLa Tet-On genome were confirmed by Southern blot. Western blots of mitochondrial extracts from MTS-EndoIII and MTS-EndoVIII clones revealed that the recombinant proteins are targeted into mitochondria and their expressions are doxycycline (Dox) dependent. Enzyme activity assays and mtDNA repair studies showed that the Dox-dependent expressions of MTS-EndoIII and MTS-EndoVIII are functional, and both MTS-EndoIII and MTS-EndoVIII (Dox+) clones were significantly more proficient at repair of oxidative damage in their mtDNA. This enhanced repair led to increased cellular resistance to oxidative stress.