The tissue inhibitor of metalloproteinase‐3 (TIMP‐3), a recently cloned member of TIMP gene family, has been implicated in the negative regulation of tumor cell invasion and tumor growth. Down‐regulation of this gene has been shown to occur in a mouse carcinogenesis model, suggesting that it might play a role in the tumor progression of some cancers. In this study, we used human gastric cancer cell lines to investigate whether TIMP‐3 gene expression is suppressed in human gastric cancer. We examined whether aberrant DNA methylation of the 5'‐CpG island of the TIMP‐3 gene is involved in this cancer. Nine of 10 human gastric cancer cell lines completely lost TIMP‐3 gene expression compared with normal samples. Southern blot analysis and bisulfite genomic sequencing revealed aberrant hypermethylation near the transcription‐start site of the TIMP‐3 gene in all cell lines lacking TIMP‐3 expression. Treatment of these cell lines with the demethylating agent 5‐aza‐2'‐deoxycytidine restored TIMP‐3 gene expression. Our results suggest that the TIMP‐3 gene is another early target of tumor‐associated aberrant DNA methylation in human gastric carcinogenesis. Consequently, genetic silencing of TIMP‐3 may lead to a more malignant and invasive phenotype in these cancer cells. Int. J. Cancer 86:632–635, 2000. © 2000 Wiley‐Liss, Inc.