DNA hypomethylation is one of the major epigenetic alterations in human cancers. We have previously shown that genes identified as hypomethylated in pancreatic cancer are expressed in pancreatic cancer cell lines, but not in normal pancreatic ductal epithelium and can be reexpressed in nonexpressing cells using 'epigenetic modifying agents' such as DNA methyltransferase inhibitors. To identify additional targets for aberrant hypomethylation in pancreatic cancer, we used oligonucleotide microarrays to screen for genes that displayed expression patterns associated with hypomethylation. This analysis identified a substantial number of candidates including previously reported hypomethylated genes. A subset of eight genes were selected for further methylation analysis, and two cancer-related genes, maspin and S100P, were found to be aberrantly hypomethylated in a large fraction of pancreatic cancer cell lines and primary pancreatic carcinomas. Combined treatment with 5-aza-2'-deoxycytidie and trichostatin A resulted in synergistic induction of maspin and S100P mRNA in MiaPaCa2 cells where both genes were methylated. Furthermore, there was an inverse correlation between methylation and mRNA expression level for maspin and S100P in a large panel of pancreatic cancer cell lines. We also found a significant difference in the methylation patterns of maspin and two previously identified hypomethylated genes (trefoil factor 2 and lipocalin 2) between pancreatic and breast cancer cell lines, suggesting cancer-type specificity for some hypomethylation patterns. Thus, our present results confirm that DNA hypomethylation is a frequent epigenetic event in pancreatic cancer, and suggest that gene expression profiling may help to identify potential targets affected by this epigenetic alteration.