Maintenance of genomic stability depends on the appropriate cellular responses to DNA damage and the integrity of the DNA repair systems. We analyzed stomach tumors with microsatellite instability (MSI) for frameshift mutations in several potential targets of the mutator phenotype involved in DNA damage-response pathways, such as the ataxia telangiectasia mutated protein-related protein (ATR)-CHK1-Cdc25c pathway, and DNA repair. High frequency of mutations was found within ATR [5 (21%) of 23], MED1 [10 (43%) of 23], hMSH3 [13 (56%) of 23], and hMSH6 [10 (43%) of 23] genes. Also, a low frequency of mutations within the CHK1 gene was detected in 9% (2 of 23) of tumors. No mutations of hMLH3, ATM, BRCA1, or NBS1 genes were detected. These results confirm ATR, MED1, and CHK1 as targets of the mutator pathway in stomach tumorigenesis, and also suggest a potential role of MED1 increasing, together with hMSH3 and hMSH6, the genomic instability in the mutator pathway as a secondary mutator. Furthermore, these results suggest that the inhibition of the ATR-CHK1 DNA damage-response pathway might be involved in the tumorigenesis of gastric cancer with microsatellite instability.