Germ line mutations in CHEK2, the gene that encodes the Chk2 serine/threonine kinase activated in response to DNA damage, have been found to confer an increased risk of some cancers. We have previously reported the presence of the common deleterious 1100delC and four rare CHEK2 mutations in inherited breast cancer. Here, we report that predictions made by bioinformatic analysis on the rare mutations indicate that two of these, delE161 (483-485delAGA) and R117G, are likely to be deleterious. We show that the proteins encoded by 1100delC and delE161 are both unstable and inefficiently phosphorylated at Thr68 in response to DNA damage, a step necessary for the oligomerization of Chk2. Oligomerization is in turn necessary for additional phosphorylation and full activation of the protein. A second rare mutation, R117G, is phosphorylated at Thr68 but fails to show a mobility shift on DNA damage, suggesting that it fails to become further phosphorylated and hence fully activated. Our results indicate that delE161 and R117G encode nonfunctional proteins and are therefore likely to be pathogenic. The findings from the biochemical analysis correlate well with predictions made by bioinformatics analysis. In addition, the results imply that these mutations, as well as 1100delC, cannot act in a dominant-negative manner to cause cancer, and tumorigenesis in association with these mutations may be due to haploinsufficiency.