HIV-1 reverse transcriptase frequently incorporates ribonucleotides into the proviral DNA in macrophages, but not in lymphocytes. The enzyme exerts an efficient ribonucleotide-terminated primer extension capacity. Furthermore, ribonucleotide-editing repair is attenuated in macrophages. Tumor suppressor p53 protein, displaying an intrinsic 3'→5' exonuclease activity, was found to be involved in efficient proofreading of base-base mismatches produced during DNA synthesis. As the presence of proofreading activity is cardinal for the DNA synthesis accuracy, it was of interest to assess whether p53 can serve as a trans-acting proofreader for HIV-1 reverse transcriptase during ribonucleotide incorporation. We investigated the potential involvement of cytoplasmic p53 in error correction during insertion of ribonucleotides into DNA by recombinant HIV-1 reverse transcriptase in a p53-proficient and deficient background. Primer extension reactions were carried out to elucidate the incorporation and removal of ribonucleotides. The biochemical studies suggest that p53 is involved in a ribonucleotide damage-associated repair mechanism through its capacity to remove preformed 3'-terminal ribonucleotides, to decrease ribonucleotide incorporation and to prevent the 3'-ribo-terminated primer extension during ongoing DNA synthesis by HIV-1 reverse transcriptase. A positive correlation exists between the presence of endogenous p53 and decrease in stable incorporation of ribonucleotides into DNA with p53-harboring lysates of HCT116 cells. p53, by preferential removal of purine over pyrimidine ribonucleotides, may affect the ribonucleotide mutation spectra produced by HIV-1 reverse transcriptase. The data implies that p53 can excise incorrect sugar in addition to base mispairs, thereby expanding the role of p53 in the repair of nucleic acids replication errors.