DLC-1 (deleted in liver cancer) gene is frequently deleted in hepatocellular carcinoma. However, little is known about the genetic status and the expression of this gene in gastric cancer. In this study, Northern and Southern analysis showed that seven of nine human gastric cancer cell lines did not express DLC-1 mRNA, but contained the DLC-1 gene. To identify the mechanism of the loss of DLC-1 mRNA expression in these cell lines, we investigated the methylation status of DLC-1 gene by using methylation-specific PCR (MSP) and Southern blot, and found that five of seven DLC-1 nonexpressing gastric cancer cell lines were methylated in the DLC-1 CpG island. Treatment with 5-aza-2'-deoxycytidine (5-Aza-dC) induced DLC-1 mRNA expression in the gastric cancer cell lines that have the methylated alleles. Studies using SNU-601 cell line with methylated DLC-1 alleles revealed that nearly all CpG sites within DLC-1 CpG island were methylated, and that the in vitro methylation of the DLC-1 promoter region is enough to repress DLC-1 mRNA expression, regardless of the presence of transcription factors capable of inducing this gene. In all, 29 of 97 (30%) primary gastric cancers were also shown to be methylated, demonstrating that methylation of the DLC-1 CpG island is not uncommon in gastric cancer. In addition, we demonstrated that DLC-1 mRNA expression was induced, and an increase in the level of acetylated H3 and H4 was detected by the treatment with trichostatin A (TSA) in two DLC-1 nonexpressing cell lines that have the unmethylated alleles. Taken together, the results of our study suggest that the transcriptional silencing of DLC-1, by epigenetic mechanism, may be involved in gastric carcinogenesis.