OBJECTIVE: Cigarette smoke extracts (CSE) could promote esophageal squamous cell carcinoma (ESCC) through upregulation of cyclooxygenase-2 (COX-2) expression. Promoter methylation mediates the transcriptional modulation of the COX-2 gene. The aim of the study was to explore whether COX-2 promoter methylation regulated COX-2 expression and functional activity in ESCC exposed to CSE. METHODS: The methylation status of COX-2 promoter in two human ESCC cell lines, EC109 and TE-1, was examined using bisulfite sequencing analysis. COX-2 mRNA and protein expression were detected by reverse-transcription polymerase chain reaction and Western blot. Prostaglandin E2 (PGE2) was examined by enzyme linked immunosorbent assay (ELISA). RESULTS: The promoter was hypermethylated in TE-1 which had a low level of COX-2 expression and was hypomethylated in EC109 with a relatively high level of COX-2 expression. Stimulation by cigarette smoke ethanol extract (EE) resulted in increased COX-2 expression in EC109, but not in TE-1. Treatment with 5-aza-2'-deoxycytidine (5-aza-DC) demethylated the promoter and upregulated COX-2 expression as well as PGE2 production in TE-1, especially followed by EE stimulation. No significant effect was observed in EC109. CONCLUSION: These findings suggest that promoter methylation may be one of the mechanisms regulating COX-2 expression in ESCC in response to stimulation of CSE.