To investigate the relationship between DNA hypomethylation and gene overexpression in pancreatic cancer, we analyzed the methylation status of a subset of 18 genes previously identified by global gene expression studies as overexpressed in pancreatic cancer tissues compared with normal pancreas. For comparison, we determined the methylation status of 14 genes not known to be overexpressed in pancreatic cancer. Methylation-specific PCR analysis revealed that 19 of these 32 genes were methylated at their 5′ CpGs in normal pancreas. We then analyzed these 19 genes for their methylation pattern in pancreatic cancers and found that all 7 of the genes (claudin4, lipocalin2, 14-3-3σ, trefoil factor2, S100A4, mesothelin, and prostate stem cell antigen) that were overexpressed in the neoplastic cells of pancreatic cancers and not expressed in normal pancreatic duct displayed a high prevalence of hypomethylation in pancreatic cancer cell lines and primary pancreatic carcinomas. By contrast, only 1 of 12 genes not overexpressed in pancreatic cancer demonstrated hypomethylation (P = 0.0002). In pancreatic cancer cell lines that retained methylation of 1 or more of the 7 aforementioned overexpressed and hypomethylated genes, treatment with 5-aza-2′-deoxycytidine or with trichostatin A, either alone or in combination, almost invariably reactivated the transcription of each of these 7 genes. These results indicate that gene hypomethylation is a frequent epigenetic event in pancreatic cancer and is commonly associated with the overexpression of affected genes.