Expression of cyclin D2 is absent in 30-70% of gastric cancers. We investigated the role of promoter hypermethylation in the transcriptional silencing of cyclin D2 in five gastric cell lines and 47 primary gastric carcinomas. CpG island methylation status of the cyclin D2 gene was studied by methylation-specific polymerase chain reaction and bisulphite sequencing. RNA and protein expression was analysed by reverse transcription-PCR and Western blot, respectively. Dense methylation of cyclin D2 was detected in three cell lines (KATOIII, AGS and NCI-N87), which also lacked cyclin D2 mRNA and protein expression. Bisulphite DNA sequencing revealed that loss of cyclin D2 expression was closely associated with the density of methylation in the promoter region. Treatment with DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine, restored the cyclin D2 expression level in methylated gastric cells. Among the 47 primary gastric cancers, cyclin D2 hypermethylation was detected in 23 (48.9%) cases. None of the 23 normal gastric biopsies from noncancer patients showed hypermethylation. Hypermethylation was associated with loss of mRNA (P&<0.001) and protein (P=0.006) expressions. Our study showed that cyclin D2 hypermethylation is associated with loss of cyclin D2 expression in a subset of gastric cancers, which may suggest an alternative gastric carcinogenesis pathway in the absence of cyclin D2 expression.