The mechanism whereby the DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) is silenced in repair-deficient (Mer-) human tumor cells is unknown. The role of methylation of the 5' CpG island in MGMT gene suppression is controversial. Although we previously showed by restriction enzyme analysis that CpG methylation in this region was associated with gene suppression, methylation at such sites was generally incomplete, suggesting heterogeneity. To clarify this issue, we have unequivocally defined the methylation status of every CpG by genomic sequencing of individual cloned copies of bisulfite-modified DNA. The region from -249 to +259 at the transcription start site was virtually methylation free in HT29 cells (Mer+), whereas in BE or HeLa S3 cells (Mer-), this region was substantially methylated in every DNA copy, with "hot spots" from -249 to -103 and from +107 to +196. Up-regulation of MGMT in HeLa S3 cells induced by 5-azacytidine was accompanied by progressive demethylation and the appearance of totally unmethylated copies of DNA. We conclude that, in Mer- cells, the MGMT promoter contains specific CpG methylation hot spots that are tightly linked to and are potential markers of gene silencing.