Tissue inhibitor of metalloproteinases-3(TIMP-3), a novel member of TIMP family genes, has been recently cloned and shown to be expressed in preneoplastic but not in neoplastic mouse JB6 epidermal cells (Sun et al. 1994 Cancer Res., 54, 11139). This down regulation of the gene appears to be attributable at least in part to alteration of gene methylation (Sun et al. 1995 J. Biol. Chem., 270, 19312). Little is known, however, about the role of TIMP-3 in human cancers. We screened several human tumor cell lines for TIMP-3 expression and found that a colon carcinoma line, DLD-1, did not express TIMP-3. If down regulation of TIMP-3 is causally related to carcinogenesis, re-expression by transfection may reverse the tumor cell phenotype. We therefore overexpressed human TIMP-3 in DLD-1 cells. TIMP-3 transfectants showed a serum-dependent growth inhibition in monolayer culture and a decreased growth potential in nude mice in a manner dependent on the level of TIMP-3 expression. A transfectant expressing a high level of active hTIMP-3 completely lost the ability to form tumors following s.c. injection into nude mice. We also tested TIMP-3 expressing cells and neocontrol TIMP-3 negative cells for their ability to grow in liquid suspension culture, since both cells grew in semi-solid soft agar. As compared to neocontrol cells, TIMP-3 overexpressors formed large aggregates, followed by cell death. This effect was not mimicked by BB94, a broad MMP inhibitor. We conclude from this study that (i) TIMP-3 overexpression in human colon carcinoma cells induces growth arrest in low serum conditions and inhibits in vivo tumor growth and (ii) the TIMP-3-induced large aggregate formation and subsequent cell death under suspension growth cannot be explained by its MMP inhibitory activity.