The enzyme O6-methylguanine-DNA methyltransferase (MGMT) protects cells from the cytotoxic and mutagenic effects of alkylating agents. Approximately 20% of tumor cell lines lack MGMT activity and are highly sensitive to alkylating agents. In established cancer cell lines, MGMT expression appears to be correlated with methylation of residues in both the promoter and the body of the gene. The effect of methylation of the MGMT promoter on gene expression and carcinogenesis in primary tumors is unknown. We investigated methylation of the MGMT promoter region in primary colorectal cancers and normal colonic mucosa. We used five methylation-sensitive restriction enzymes (BssHII, SacII, Eagl, Nael, and Smal) and Southern blot analysis to assess methylation in 46 cancers and 22 controls. Methylation of Eagl and Nael sites was seen in 12 tumors but in none of the 22 normal colorectal mucosa specimens. This difference was statistically significant (P<0.01). Methylation-sensitive single-nucleotide primer extension analysis of four additional cytosine residues confirmed methylation of the promoter region in the tumors identified by Eagl and Nael digestions and served to further quantitate the extent of methylation. Western blot analysis of 21 tumors revealed statistically significant lower MGMT expression in the eight tumors with methylation of the Eagl and Nael sites and nt -128 than in the 13 tumors lacking the methylation pattern (P<0.05). MGMT activity was lower in tumors with methylation than in tumors that were not methylated. The difference was not, however, statistically significant. We conclude that a subset of colorectal tumors is characterized by a specific methylation pattern in the MGMT promoter associated with reduced MGMT expression.