The checkpoint kinase 2 (CHEK2, also known as CHK2) is a tumor suppressor that participates in the DNA damage-signaling pathway. It is phosphorylated and activated following DNA damage, resulting in cell cycle arrest and apoptosis. Previously, we reported germlineCHEK2mutations in patients with prostate cancer. In this study, we have identified two novel somaticCHEK2mutations, c.349A>G (p.R117G) and c.967A>C (p.E321K), in prostate tumor specimens and investigated the functions of these mutants in vivo. We have shown that most of the germlineCHEK2mutations and one somatic mutation (p.R117G) within FHA domain have modestly reduced CHEK2 kinase activity in comparison with wild-typeCHEK2while the other somatic mutation (p.E321K) within the kinase domain of CHEK2 totally abolished CHEK2 kinase activity. Given that several clinical CHEK2 mutations reside in the Forkhead-associated (FHA) domain, we further generated a series of missense mutations within this domain and demonstrated the requirement of an intact FHA domain for the full activation of CHEK2. Taken together, these results provide evidence that both germline and somaticCHEK2mutations identified in prostate cancer may contribute to the development of prostate cancer through the reduction of CHEK2 activation in response to DNA damage and/or oncogenic stress. Hum Mutat 27(8), 742–747, 2006. Published 2006 Wiley-Liss, Inc.