Microsatellite unstable cancers account for up to 15% of sporadic colon cancers and are predominantly located in the proximal colon. These cancers commonly show MLH1 promoter methylation and the CpG island methylator phenotype (CIMP). A potential precursor of sporadic unstable cancers, the proximal hyperplastic polyp, is also reported to have CIMP and MLH1 methylation. However, this latter finding is not supported by MLH1 protein expression studies. To help resolve this apparent discrepancy, we determined MLH1 promoter methylation and CIMP by quantitative real-time PCR for 29 proximal hyperplastic polyps, 23 distal hyperplastic polyps, and 11 sporadic microsatellite unstable colon cancers. BRAF V600E mutation status was also determined. Positive methylation was defined as the percentage of methylated reference (PMR) >10. Only 1 of 29 proximal hyperplastic polyps showed positive MLH1 methylation (PMR of 13.0). Neither this polyp nor seven other proximal polyps with PMR values between 0 and 10 showed loss of MLH1 protein expression by immunohistochemistry. In contrast, all 11 microsatellite unstable cancers showed high degrees of MLH1 methylation, with PMR values >30. Fourteen of twenty-nine (48%) of the proximal hyperplastic polyps and 1 of 23 (4%) of the distal hyperplastic polyps showed CIMP (P<0.001). Of the unstable cancers, 10 of 11 showed CIMP. The PMR values in the CIMP-positive proximal hyperplastic polyps were significantly lower than those of the unstable cancers for 4 of the 5 CIMP markers (P<0.05). BRAF V600E mutations were seen in 83% of proximal and 74% of distal hyperplastic polyps. Quantitative analysis of MLH1 methylation does not support earlier reports of MLH1 methylation in proximal hyperplastic polyps. However, these lesions do harbor promoter methylation at other CIMP loci, although at a lower level than that seen in unstable cancers. If these polyps are the precursor for sporadic microsatellite unstable cancers, then MLH1 methylation and higher degrees of promoter methylation in general occur at a later stage of carcinogenesis.