Mutational inactivation of hMSH2 or hMLH1 has been known to be responsible for microsatellite instability and cellular resistance to DNA-damaging alkylating agents. However, the effects of altered expression of hMSH2 or hMLH1 on microsatellite stability and cellular response to alkylating agents has not been well investigated. Previously, we have reported that downregulation of the hMLH1 protein was a frequent event and was closely associated with cellular resistance to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in human gastric carcinoma cell lines. Therefore, to investigate the relationship between microsatellite instability and quantitative changes in hMSH2 and hMLH1, we compared the genetic status and expression levels of hMSH2 and hMLH1 with microsatellite instability in 11 human gastric carcinoma cell lines. Five cell lines contained wild-type hMSH2 and hMLH1 and expressed adequate levels of hMSH2 and hMLH1 proteins. In three cell lines, genetic alterations such as mutation in the hMLH1 gene (SNU-1) or the hMSH2 gene (SNU-638), or hypermethylation in the promoter region of the hMLH1 gene (SNU-520) were observed. Microsatellite instability assays revealed that only these three cell lines exhibited microsatellite instability. Three cell lines (SNU-216, -484, and -668) containing wild-type hMSH2 and hMLH1 genes produced significantly downregulated hMSH2 and/or hMLH1 proteins. In spite of the substantial decrease in the protein levels, these cell lines did not show microsatellite instability. Together with our previous report, this study suggests that: microsatellite instability of cells is associated only with genetic alteration of the mismatch repair genes; relatively low levels of the hMSH2 and hMLH1 proteins may be sufficient to retain the microsatellite stable phenotype; and the cellular response to alkylating agents is associated with genetic alteration and decreased expression of the mismatch repair genes in human gastric carcinoma cell lines.