The tumor suppressor p53 plays a critical role in the regulation of the cell cycle and the maintenance of genetic stability. The 3'-->5' exonuclease activity of p53 has recently been recognized as a novel biochemical function of this molecule, but the biological significance of this activity remains elusive. Using an in vitro DNA replication assay with purified human DNA polymerases, p53 protein, and defined DNA primer/templates, we demonstrated that the wild-type (wt) p53 protein, but not the mutant p53 protein, specifically enhanced the DNA replication fidelity of polymerase (pol) alpha, an enzyme that lacks 3'-->5' exonuclease activity. The misincorporation of non-complementary deoxynucleotides into DNA by pol alpha was substantially decreased by p53. In contrast, wt p53 showed no significant effect on replication fidelity or the rates of DNA synthesis by human pol epsilon or the bacterial enzyme pol I. Inhibition of 3'-->5' exonuclease activity by guanosine monophosphate (GMP) abolished the ability of p53 to enhance the replication fidelity of pol alpha. Quantitative analyses revealed that the 3'-->5' exonuclease activity of p53 effectively removed mismatched nucleotides from DNA in preference over matched nucleotides. Furthermore, study in intact cells revealed that the wt p53 protein was co-localized with DNA synthesis activity in S phase cells. These results suggest a possibility that the 3'-->5' exonuclease of the wt p53 protein might provide the proof-reading function for DNA pol alpha. The preferential excision of mismatched nucleotides from the replicating DNA strand appears to be a potential biochemical mechanism by which p53 enhances DNA replication fidelity and thereby helps to maintain genomic integrity.