The tumor suppressor p53 plays an important role in response to DNA damage, including DNA repair. One DNA repair pathway, nucleotide excision repair (NER), has been well-documented to be regulated by p53. It seemed probable that p53 may affect other DNA repair pathways. We employed matched isogenic pairs of cell lines, wild-type or p53-deficient, to investigate this question using methyl methanesulfonate (MMS), a base-damaging agent. Alkylation damage induced by MMS is repaired exclusively by the base excision repair (BER) pathway. Cells carrying mutant or no p53 genes exhibited slow BER of MMS-induced DNA damage, and exhibited MMS-sensitivity. One contributing factor is the abundance of DNA polymerase beta (beta-pol), an enzyme required for BER, which was almost absent in p53 mutant and p53-null cells. Our findings demonstrate an in vivo requirement for p53 in regulating the base excision repair response, a novel finding of great potential importance in understanding the DNA repair branch of the p53 pathway.