In response to DNA damage, cells transduce a signal that leads to accumulation and activation of p53 protein, transcriptional induction of several genes, including p21, gadd45, and gadd153, and cell cycle arrest. One hypothesis is that the signal is mediated by DNA-dependent protein kinase (DNA-PK), which consists of a catalytic subunit (DNA-PKcs) and a regulatory subunit (Ku). DNA-PK has several characteristics that support this hypothesis: Ku binds to DNA damaged by nicks or double-strand breaks, DNA-PKcs is activated when Ku binds to DNA, DNA-PK will phosphorylate p53 and other cell cycle regulatory proteins in vitro, and DNA-PKcs shares homology with ATM, which is mutated in ataxia telangiectasia and involved in signaling the p53 response to ionizing radiation. The hypothesis was tested by analyzing early passage fibroblasts from severe combined immunodeficient mice, which are deficient in DNA-PK. After exposure to ionizing radiation, UV radiation, or methyl methane-sulfonate, severe combined immunodeficient and wild-type cells were indistinguishable in their response. The accumulation of p53, induction of p21, gadd45, and gadd153, and arrest of the cell cycle in G1 and G2 occurred normally. Therefore, DNA-PK is not required for the p53 response or cell cycle arrest after DNA damage.