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Previous studies on esophageal squamous cell carcinoma (ESCC) indicated that it contains much dysregulation of microRNAs (miRNAs). DNA hypermethylation in the miRNA 5′ regulatory region is a mechanism that can account for the downregulation of miRNA in tumors (Esteller, N Engl J Med 2008;358:1148–59). Among those dysregulated miRNAs, miR-203, miR-34b/c, miR-424 and miR-129–2 are embedded in CpG islands, as is the promoter of miR-34a. We investigated their methylation status in ESCC by bisulfite sequencing PCR (BSP) and methylation specific PCR (MSP). The methylation frequency of miR-203 and miR-424 is the same in carcinoma and in the corresponding non-tumor tissues. The methylation ratio of miR-34a, miR-34b/c and miR-129–2 is 66.7% (36/54), 40.7% (22/54) and 96.3% (52/54), respectively in ESCC, which are significantly higher than that in the corresponding non-tumor tissues(p < 0.01). Quantitative RT-PCR analysis in clinical samples suggested that CpG island methylation is significantly correlated with their low expression in ESCC, 5-aza-2′-deoxycytidine (DAC) treatment partly recovered their expression in EC9706 cell line. We conclude that CpG island methylation of miR-34a, miR-34b/c and miR-129–2 are frequent events and important mechanism for their low expression in ESCC. DNA methylation changes have been reported to occur early in carcinogenesis and are potentially good early indicators of carcinoma (Laird, Nat Rev Cancer 2003;3:253–66). The high methylation ratio of miR-129–2 indicated its potential as a methylation biomarker in early diagnosis of ESCC.