Microsatellite instability (MSI) has been described in tumors from patients with hereditary nonpolyposis colorectal cancer, sporadic colorectal cancer, and other types of cancers. MSI is caused by the dysfunction of mismatch repairs genes. Loss of expression and mutation in one of the major mismatch repair genes, hMLH1, and the methylation of CpG sites in its promoter occur frequently in primary tumors and cell lines of colorectal cancer with MSI. To understand the mechanisms involved in the silencing of hMLH1 expression by methylation, we examined the methylation status of all CpG sites in the hMLH1 promoter in 24 colorectal cancer cell lines by the NaHSO3-sequencing method. We identified a small proximal region (-248 to -178, relative to the transcription start site) in the promoter in which the methylation status invariably correlates with the lack of hMLH1 expression. This correlation was further supported by the observation that cell lines that showed methylation-suppressed hMLH1 expression can be induced to reexpress hMLH1 by a methyl transferase inhibitor, 5-aza-2'-deoxycytidine, and the small region that we identified exhibited significant demethylation in all cell lines examined.