Matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases (TIMPs) regulate the structural integrity of the extracellular matrix (ECM). Constitutive expression of human TIMP-3 in human DLD colon carcinoma cells renewed serum-responses and inhibited tumour formation in nude mice. To elucidate the mechanism of TIMP-3-mediated tumour suppression, we compared parental DLD and TIMP-3 expressing DLD cells (TIMP-3/DLD), finding them to be significantly different. TIMP-3/DLD cultures have fewer mitotic cells, are delayed in G₁, and die after serum starvation. TIMP-3/DLD conditioned media activates cell death on fibroblast cells. The cell death induced by serum starvation and conditioned media was inhibited by 70%, in the presence of neutralizing tumour necrosis factor α (TNF-α) antibody. TIMP-3/DLD whole cell lysate contained p55 TNF-α receptor, while vector/DLD lysate had p55 TNF-α receptor and p46 soluble TNF-α inhibitor. Vector/DLD conditioned media had p46, while no soluble TNF-α receptor was detected in TIMP-3/DLD conditioned media. In addition, FACS analysis revealed that TIMP-3/DLD cells have more TNF-α surface binding sites, suggesting a direct correlation between TIMP-3 expression and surface receptors. The mechanism of tumorigenic reversion induced by TIMP-3 in DLD cells may involve protection of receptors from the proteolytic activity of MMPs. Putative TIMP-3-mediated inhibition of MMPs restores the TNF-α p55 signalling pathway and the carcinoma cell is killed by autocrine TNF-α. Thus, DLD cells have specific ECM MMPs that cleave cytokines and cytokine receptors. TIMP-3 specifically inhibits MMPs involved in receptor shedding.