Cyclooxygenase-2 (COX-2) is overexpressed in various types of human malignancies including esophageal squamous cell carcinoma (ESCC). However, a subset of ESCC either do not express COX-2 or show low level of expression. It is well established that promoter methylation is a major mechanism that mediates transcriptional silencing of COX-2 in gastric and colorectal cancer, but the data on ESCC are very limited. In this study, we attempted to determine whether COX-2 expression was also regulated by promoter methylation in human ESCC cell lines. We examined the methylation status of the COX-2 promoter in five human ESCC cell lines (EC109, EC9706, KYSE 410, KYSE 150, TE-1) using bisulfite sequencing analysis. Western blot analysis was used to determine COX-2 expression. Quantitative real-time polymerase chain reaction was used to determine COX-2 mRNA level. Prostaglandin (PG) E-2 was detected by ELISA. The promoter was densely methylated in TE-1 and KYSE 150, which had a low level of COX-2 expression and less methylated in other three cell lines (EC109, EC9706, KYSE 410), with high level of COX-2 expression. Treatment with 5-aza-deoxycytidine (5-aza-DC), a DNA methyltransferase inhibitor, demethylated the promoter and upregulated COX-2 expression, as well as PGE(2) production in TE-1 and KYSE 150. However, no such effects were observed in EC109. COX-2 protein was negative, but mRNA was positive in TE-1. After treatment with 5-aza-DC, both COX-2 mRNA and protein level had increased. These findings suggest that the promoter methylation may be one of the mechanisms that regulate COX-2 expression in ESCC.