The tumor suppressor p53 protein plays a critical role in the cell-cycle progression. The role of the 3'-to-5' exonuclease activity of p53 protein in the DNA repair process remains elusive. Using an in vitro exonuclease assay and defined oligonucleotides terminated with beta-D- and beta-L-nucleoside analogs at the 3'-terminus, we studied the ability of p53 protein to excise beta-L- and beta-D-nucleoside analogs which have anticancer or antiviral potential. p53 protein removes beta-D-nucleoside analogs more efficiently compared to that of beta-L-nucleoside analogs. The affinity of p53 protein for an beta-L-nucleotide terminated primer was 5 fold lower compared to non-modified primer. The hypothesis on an important role of the 3'-to-5' exonuclease activity of p53 protein in the action of nucleoside analogs was proposed.