The murine Chk2 kinase is activated after exposure to ionizing radiation and is necessary for p53-dependent apoptosis, but the role Chk2 plays in determining genomic stability is poorly understood. By analyzing the sensitivity of Chk2-deficient murine and human cells to a range of DNA-damaging agents, we show that Chk2 deficiency results in resistance to agents that generate double-strand breaks but not to other forms of damage. Surprisingly, the absence of Chk2 results in increased sensitivity to UV-radiation-induced DNA damage. Defective apoptosis after radiation-induced DNA damage may result in genomic instability; therefore, the consequences of Chk2 deficiency on genomic instability were assayed using an in vitro screen. Gene amplification was not detected in untreated Chk2(-/-) cells, but the rate of gene amplification after irradiation was elevated and was similar to that found in p53 compromised cells. A synergistic increase in genomic instability was seen after disruption of both Chk2 and p53 function, indicating that the two proteins have non-redundant roles in regulating genome stability after irradiation. The data demonstrate that Chk2 functions to maintain genome integrity after radiation-induced damage and has important implications for the use of Chk2 inhibitors as adjuvant cancer therapy.