DNA polymerase beta (Polbeta) has been implicated in base excision repair. Polbeta knockout mice exhibit apoptosis in postmitotic neuronal cells and die at birth. Also, mice deficient in nonhomologous end-joining (NHEJ), a major pathway for DNA double-strand break repair, cause massive neuronal apoptosis. Severe combined immunodeficiency (SCID) mice have a mutation in the gene encoding DNA-dependent protein kinase catalytic subunit (DNA-PKcs), the component of NHEJ, and exhibit defective lymphogenesis. To study the interaction between Polbeta and DNA-PKcs, we generated mice doubly deficient in Polbeta and DNA-PKcs. Polbeta(-/-)DNA-PKcs(scid/scid) embryos displayed greater developmental delay, more extensive neuronal apoptosis, and earlier lethality than Polbeta(-/-) and DNA-PKcs(scid/scid) embryos. Furthermore, to study the involvement of p53 in the phenotype, we generated Polbeta(-/-)DNA-PKcs(scid/scid)p53(-/-) triple-mutant mice. The mutants did not exhibit apoptosis but were lethal with defective neurulation at midgestation. These results suggest a genetic interaction between Polbeta and DNA-PKcs in embryogenesis and neurogenesis.