Hypermethylation of the hMLH1 promoter is observed in the majority of sporadic gastric carcinomas with high frequency microsatellite instability (MSI), and it contributes to the genesis of MSI-positive gastric carcinoma. Multiple gastric carcinoma is known to have a higher frequency of MSI positivity than single gastric carcinoma. However, the molecular basis of MSI in these tumors remains obscure. We investigated the role of hMLH1 promoter hypermethylation in the genesis of multiple gastric carcinoma with MSI. We analyzed 33 tumors from 15 patients with multiple gastric carcinoma (12 double tumors and three triple tumors) for MSI, expression of hMLH1 and hMSH2, and hypermethylation of hMLH1 and hMSH2 promoters. High frequency MSI was found in seven out of 33 tumors (21%) in five out of 15 patients (33%). All of the tumors with high frequency MSI had a lack of hMLH1 expression, with the presence of hMSH2 expression, while all the tumors with no MSI or low frequency MSI were positive for both hMLH1 and hMSH2. All of the tumors with no expression of hMLH1 had hMLH1 hypermethylation, whereas hMLH1 hypermethylation was observed in two out of 26 (8%) tumors with no or low frequency MSI. None of the tumors showed hMSH2 hypermethylation. These results suggest that epigenetic changes in the hMLH1 promoter account for the genesis of multiple gastric carcinoma with high frequency MSI.