The mitochondrial respiratory chain is a source of reactive oxygen species (ROS) that are responsible for oxidative modification of biomolecules, including proteins. Due to its association with mitochondrial DNA, DNA polymerase γ (pol γ) is in an environment to be oxidized by hydrogen peroxide and hydroxyl radicals that may be generated in the presence of iron ions associated with DNA. We tested whether human pol γ was a possible target of ROS with H ₂ O ₂ and investigated the effect on the polymerase activities and DNA binding efficiency. A 1 h treatment with 250 µM H ₂ O ₂ significantly inhibited DNA polymerase activity of the p140 subunit and lowered its DNA binding efficiency. Addition of p55 to the p140 catalytic subunit prior to H ₂ O ₂ treatment offered protection from oxidative inactivation. Oxidatively modified amino acid residues in pol γ  resulting from H ₂ O ₂ treatment were observed in vitro as well as in vivo , in SV40-transfected human fibroblasts. Pol γ was detected as one of the major oxidized mitochondrial matrix proteins, with a detectable decline in polymerase activity. These results suggest pol γ as a target of oxidative damage, which may result in a reduction in mitochondrial DNA replication and repair capacities.