The mammalian Chk2 kinase is thought to mediate ATM-dependent signaling in response to DNA damage. The physiological role of mammalian Chk2 has now been investigated by the generation of Chk2-deficient mice. Although Chk2(-/-) mice appeared normal, they were resistant to ionizing radiation (IR) as a result of the preservation of splenic lymphocytes. Thymocytes and neurons of the developing brain were also resistant to IR-induced apoptosis. The IR-induced G(1)/S cell cycle checkpoint, but not the G(2)/M or S phase checkpoints, was impaired in embryonic fibroblasts derived from Chk2(-/-) mice. IR-induced stabilization of p53 in Chk2(-/- )cells was 50-70% of that in wild-type cells. Caffeine further reduced p53 accumulation, suggesting the existence of an ATM/ATR-dependent but Chk2-independent pathway for p53 stabilization. In spite of p53 protein stabilization and phosphorylation of Ser23, p53-dependent transcriptional induction of target genes, such as p21 and Noxa, was not observed in Chk2(-/-) cells. Our results show that Chk2 plays a critical role in p53 function in response to IR by regulating its transcriptional activity as well as its stability.