The p53 gene regulates the cell cycle response to DNA damage, which may allow time for adequate DNA repair. We asked whether p53 could directly increase the repair of defined double-strand breaks (DSBs) by nonhomologous end-joining in gamma-irradiated mouse embryonic fibroblasts with differing p53 status. By using an episomal plasmid reactivation assay, we found that presence of wild-type p53 enhanced rejoining of DSBs with short complementary ends of single-stranded DNA. p53 appeared to be directly involved in this regulation, because rejoining enhancement was dependent on the presence of nonspecific DNA binding activity as mediated by the COOH-terminal domain and was independent of transactivating function. We hypothesize that tumor cells lacking p53 and normal cells with wild-type p53 may use different pathways for repair of radiation-induced DSBs.