The TSLC1 (tumor suppressor in lung cancer-1) gene is a novel tumor suppressor gene on chromosomal region 11q23.2, and is frequently inactivated by concordant promoter hypermethylation and loss of heterozygosity (LOH) in non-small cell lung cancer (NSCLC). Because LOH on 11q has also been observed frequently in other human neoplasms including gastric cancer, we investigated the promoter methylation status of TSLC1 in 10 gastric cancer cell lines and 97 primary gastric cancers, as well as the corresponding non-cancerous gastric tissues, by bisulfite-SSCP analysis followed by direct sequencing. Allelic status of the TSLC1 gene was also investigated in these cell lines and primary gastric cancers. The TSLC1 promoter was methylated in two gastric cancer cell lines, KATO-III and ECC10, and in 15 out of 97 (16%) primary gastric cancers. It was not methylated in non-cancerous gastric tissues, suggesting that this hypermethylation is a cancer-specific alteration. KATO-III and ECC10 cells retained two alleles of TSLC1, both of which showed hypermethylation, associated with complete loss of gene expression. Most of the primary gastric cancers with promoter methylation also retained heterozygosity at the TSLC1 locus on 11q23.2. These data indicate that bi-allelic hypermethylation of the TSLC1 promoter and resulting gene silencing occur in a subset of primary gastric cancers.