The mutation rate of the mammalian mitochondrial genome is higher than that of the nuclear genome. Because mitochondrial and nuclear deoxyribonucleoside triphosphate (dNTP) pools are physically distinct and because dNTP concentrations influence replication fidelity, we asked whether mitochondrial dNTP pools are asymmetric with respect to each other. We report here that the concentrations of the four dNTPs are not equal in mitochondria isolated from several tissues of both young and old rats. In particular, in most tissues examined, mitochondrial dGTP concentrations are high relative to the other dNTPs. Moreover, in the presence of the biased dNTP concentrations measured in heart and skeletal muscle, the fidelity of DNA synthesis in vitro by normally highly accurate mtDNA polymerase gamma is reduced, with error frequencies increased by as much as 3-fold, due to increased formation of template T.dGTP mismatches that are inefficiently corrected by proofreading. These data, plus some published data on specific mitochondrial mutations seen in human diseases, are consistent with the hypothesis that normal intramitochondrial dNTP pool asymmetries may contribute to spontaneous mutagenesis in the mammalian mitochondrial genome.