The first-degree relatives of patients affected by colorectal cancer, who do not belong to familial adenomatous polyposis and hereditary nonpolyposis colorectal cancer families, have a doubled risk of developing tumors of the large intestine. We have previously demonstrated that subjects with a single first-degree relative (SFDR) with colon cancer have a doubled risk for developing colorectal adenomas, and in these cases, polyps recur more frequently. The mechanism underlying this predisposition has not been clarified. In this study, we evaluated the frequency of microsatellite instability (MSI) using the five markers suggested by the National Cancer Institute workshop, target gene mutations, hMLH1 and hMSH2 expression, and hMLH1 promoter hypermethylation in the adenomas of patients with and without a SFDR affected by colon cancer. Seventy polyps were obtained from 70 patients: 27 with a single FDR with colon cancer and 43 without such a history. Of the 70 polyps, 12 were MSI-H (17.1%), 20 were MSI-L (28.6%), and 30 were microsatellite stable (42.9%). Of the 27 patients with positive family history, 8 polyps (29.6%) were MSI-H compared with those with negative history in which 4 polyps (9.3%) were MSI-H (P < 0.02). Of the 12 MSI-H polyps, all of the polyps obtained from patients with positive family history had loss of hMLH1 immunostaining versus one with negative family history (P < 0.02). Of the MSI-H polyps, 2 had a somatic frameshift mutation of the MBD4 gene, 1 of MSH6, 1 of BAX, and 2 of transforming growth factor betaRII. Furthermore, 6 of 8 polyps from patients with positive family history with MSI-H and loss of MLH1 had hypermethylation of the MLH1promoter versus none of the MSI-H with negative family history (P < 0.02). All 6 polyps of the 27 from SFDR positive subjects, with hMLH1 promoter hypermethylation loss of hMLH1 and MSI, were located in the right colon (P < 0.02). Hypermethylation of the promoter of hMLH1, consequent loss of hMLH1 expression, and MSI are at the basis of approximately 25% of adenomatous polyps developed in subjects with a SFDR affected by colorectal cancer.