The human mismatch repair (MMR) gene MSH2 is the second most frequently mutated hereditary nonpolyposis colorectal cancer (HNPCC) susceptibility locus. Given that missense mutations account for 17% of all identified alterations in this gene, the study of their pathogenicity is of increasing importance. Previously, we showed that pathogenic MSH2 missense mutations typically impaired the repair activity of the protein. In this study, we took advantage of its crystal structure and attempted to correlate the mismatch binding and ATP-catalyzed mismatch release activities with the location of 18 nontruncating MSH2 mutations. We observed that the MMR-deficient mutations situated in the amino-terminal connector and lever domains of MSH2 (V161D, G162R, G164R, L173P, L187P, C333Y, and D603N) affected protein stability, whereas mutations in the ATPase domain (A636P, G674A, C697F, I745_I746del, and E749 K) mainly caused defects in mismatch binding or release. Of the MMR-proficient variants, four (T33P, A272 V, G322D, and V923E) showed slightly reduced mismatch binding and/or release efficiencies compared to wild-type (WT) protein, while two variants (N127S and A834 T) showed no defects in the assays. Similar to our biochemical data, the mutations that affected protein stability were associated with an absence of the protein in tumors in immunohistochemical (IHC) analyses. In contrast, the protein with the mutation E749 K, which abrogates MMR but not protein stability, is well expressed in tumors. In conclusion, pathogenic missense mutations in MSH2 may interfere with different mechanisms that tend to cluster in separate protein domains with varying effects on protein stability, which could be taken into account when interpreting IHC data.