The tumor suppressor p53 plays a central role in the DNA damage response. p53 enhances base excision repair (BER), in part, through direct interaction with the repair complex. Mitochondrial DNA (mtDNA) is repaired by a mtBER pathway. Many colorectal cancers harbor mtDNA mutations that are associated with poor prognosis. In addition to modulating the apoptotic response, mitochondria-localized p53 also stimulates mtBER. However, the mechanisms by which p53 enhances colorectal cancer mtBER after stress remain unclear. To explore this, we used colorectal cancer cells isogenic for p53 (HCT116p53+/+ and HCT116p53-/-). p53+/+ cells more efficiently repaired H(2)O(2) damaged DNA in vivo as measured by semiquantitative mtDNA displacement loop PCR. Mitochondrial extracts from p53+/+ cells more efficiently stimulated (32)P-dCTP incorporation into a uracil-oligonucleotide. Recombinant p53 complemented p53-/- mitochondrial extract repair of uracil or 8-oxo-G-containing oligonucleotides. As a measure of DNA glycosylase activity, p53+/+ mitochondrial extracts more efficiently incised uracil or 8-oxo-G oligonucleotides, although recombinant p53 could not stimulate oligonucleotide incision. p53 did not influence mitochondrial apurinic/apyrimidinic endonuclease activity measured by incision of a tetrahydrofuran-oligonucleotide. p53+/+ mitochondrial extracts had higher DNA polymerase-gamma activity measured by (32)P-dCTP incorporation into a single-nucleotide gap oligonucleotide, and recombinant p53 complemented p53-/- mitochondrial extract DNA polymerase-gamma activity. mtDNA ligase activity was not affected by p53 status. p53 protein was detected in an inner mitochondrial membrane subfraction containing components of the mtBER complex. Our data suggest that an intact p53 pathway stimulates specific mtBER steps and provides mechanistic insight into the development of mtDNA mutations in colorectal cancer.