The tumor suppressor protein p53 displays 3' --> 5' exonuclease activity and can provide a proofreading function for DNA polymerases. Reverse transcriptase (RT) of human immunodeficiency virus (HIV)-1 is responsible for the conversion of the viral genomic ssRNA into the proviral DNA in the cytoplasm. The relatively low fidelity of HIV-1 RT was implicated as a dominant factor contributing to the genetic variability of the virus. The lack of intrinsic 3' --> 5' exonuclease activity, the formation of 3'-mispaired DNA and the subsequent extension of this DNA were shown to be determinants for the low fidelity of HIV-1 RT. It was of interest to analyse whether the cytoplasmic proteins may affect the accuracy of DNA synthesis by RT. We investigated the fidelity of DNA synthesis by HIV-1 RT with and without exonucleolytic proofreading provided by cytoplasmic fraction of LCC2 cells expressing high level of wild-type functional p53. Two basic features related to fidelity of DNA synthesis were studied: the misinsertion and mispair extension. The misincorporation of noncomplementary deoxynucleotides into nascent DNA and subsequent mispair extension by HIV-1 RT were substantially decreased in the presence of cytoplasmic fraction of LCC2 cells with both RNA/DNA and DNA/DNA template-primers with the same target sequence. The mispair extension frequencies obtained with the HIV-1 RT in the presence of cytoplasmic fraction of LCC2 cells were significantly lower (about 2.8-15-fold) than those detected with the purified enzyme. In addition, the productive interaction between polymerization (by HIV-1 RT) and exonuclease (by p53 in cytoplasm) activities was observed; p53 preferentially hydrolyses mispaired 3'-termini, permitting subsequent extension of the correctly paired 3'-terminus by HIV-1 RT. The data suggest that p53 in cytoplasm may affect the accuracy of DNA replication and the mutation spectra of HIV-1 RT by acting as an external proofreader. Furthermore, the decrease in error-prone DNA synthesis with RT in the presence of external exonuclease, provided by cytoplasmic p53, may partially account for lower mutation rate of HIV-1 observed in vivo.