CpG island hypermethylation is a potential means of inactivating tumor suppressor genes, and many genes have been demonstrated to be hypermethylated and silenced in colorectal cancer. However, limited data is available upon the concurrent methylation of multiple genes in colorectal cancer and in its precursor lesion. To address changes in the methylation profiles of multiple genes during colorectal carcinogenesis, we investigated the methylation of 12 genes (APC, COX-2, DAP-kinase, E-cadherin, GSTP1, hMLH1, MGMT, p14, p16, RASSF1A, THBS1, and TIMP3) in normal colon (n ¼ 24), colon adenoma (n ¼ 95), and colorectal cancer (n ¼ 149), using methylation-specific PCR. The average number of these genes methylated per sample was 0.12, 1.8, and 3.0 in normal colon mucosa, adenoma, and carcinoma, respectively, showing a stepwise increase (Po0.001). All the genes were methylated in colorectal cancer at frequencies varying from 51 to 9.4% and colon adenoma displayed methylation for the 11 genes, except for GSTP1, at frequencies varying from 40 to 1.1%. In contrast, normal colon mucosa demonstrated methylation for APC only, at a frequency of 12.5%. The total number of methylated genes per tumor showed a continuous, nonbimodal distribution in colon adenoma or cancer. CpG island hypermethylation exhibited a proclivity toward proximal colon cancer or adenoma, and the average number of genes methylated was higher in proximal colon cancer or adenoma than in distal colon cancer or adenoma, respectively (3.5 vs 2.6, P ¼ 0.018 for cancer, and 2.5 vs 1.4, P ¼ 0.003 for adenoma).In conclusion, concurrent CpG island methylation is an early and frequent event during colorectal carcinogenesis. It appears that CpG island methylation plays a more important role in proximal colon cancer development than in distal colon cancer development. Keywords: CpG island; colon adenoma; colorectal cancer; DNA methylation; tumor suppressor genes There are two well-known pathways of colorectal carcinogenesis, that is, chromosomal instability (CIN) and microsatellite instability (MSI). The CIN pathway is characterized by alterations of chromosomal number and structure affecting protooncogenes and tumor suppressor genes. 1 The CIN phenotype shows a high frequency of allelic losses and abnormal DNA content by flow cytometry. 2 Whereas, the MSI pathway features alterations in repeated nucleotides within the coding sequences, which result in the inactivation of tumor suppressor genes. 3,4 In contrast to the CIN phenotype, the MSI phenotype shows near-diploidy and very low allelic loss frequencies. 2 Recently the CpG island methylation phenotype has been added to these two phenotypes, and is characterized by the concordant methylation of the promoter regions of multiple genes that play a role in carcinogenesis. 5 Moreover, accumulating evidence has now linked promoter CpG island methylation with transcriptional silencing in human cells. Although the mechanism whereby CpG island methylation suppresses gene transcription has not been fully elucidated, it has bee...