The DNA mismatch repair (MMR) pathway is essential in maintaining genomic stability through its role in DNA repair and the checkpoint response. Loss of DNA MMR underlies the hereditary cancer disease Lynch Syndrome (LS). Germline mutations in MSH2 account for approximately 40% of LS patients and of these, 18% are missense variants. One important clinical challenge has been discriminating between missense variants that are pathogenic and those that are not. Current analysis of missense mutations in MSH2 is performed using a combination of clinical, biochemical, and functional data; however, suitable cell culture models to test the various functions of the DNA MMR proteins are lacking. Here, we have generated human cell lines stably expressing a subset of MSH2 missense mutants and tested their effect on DNA repair and checkpoint response functions. We have expanded on previous biochemical and functional analyses performed in non-human systems to further understand defects conferred by this subset of single amino acid alterations. The functional characterization of MSH2 missense mutants combined with clinical and biochemical data is essential for appropriate patient management and genetic counseling decisions. ©2010 Wiley-Liss, Inc.