The tumor suppressor protein p53 plays an important role in maintaining genomic integrity. p53 possesses an intrinsic 3→ 5 exonuclease activity. The presence of exonuclease activity has been implicated in the prevention of mutator phenotype and in acquired resistance to chemotherapeutic agents. p53 may act as proofreading exonuclease for DNA polymerases during DNA replication. The recombinant wild-type p53 can proofread for various exonuclease-deficient cellular and viral DNA polymerases. The preferential excision of mismatched nucleotides from DNA by 3→5 exonuclease activity enhances the fidelity of DNA synthesis, thus providing a biochemical mechanism to reduce mutations caused by incorporation of mismatched nucleotides. P53 exonuclease activity for different mismatches is dependent upon the nature of the mispair. The observed specificity of mismatch excision indicates that p53 exonucleolytic proofreading preferentially repairs transversion mutations. Interestingly, p53 exonuclease activity preferentially removes the same base that is less efficiently incorporated and extended by the DNA polymerase. Hence, the proofreading activity of p53 may limit the transversion mutations, indicating that the mutation spectra might be affected by the actions of DNA polymerase and p53-exonuclease. p53 was found in the nucleus and in the cytoplasm of the cell. In cytoplasmic extracts, non-induced p53, displays high level of 3→ 5 exonuclease activity in comparison to nuclear extracts. P53 may recognize and remove incorporated nucleoside analogs from DNA in vitro, in whole cells and in cytoplasm. These findings raise the possibility that resistance to anti-cancer and anti-viral nucleoside analogs, in part, may involve the excision of incorporated drug during DNA synthesis.