Matrix metalloproteinases (MMPs) are essential for extracellular matrix (ECM) breakdown and repair, and have been implicated in the development of metastases. TIMP-3 was initially identified as a potent inhibitor of MMPs, however it also has several properties that are unique and not related to its ability to abrogate MMPs. We studied the effects of overexpression of tissue inhibitor of metalloproteinases-3 (TIMP-3) on lung cancer cells and explored the mechanisms involved in apoptosis-induction in susceptible cells and subsequently, the therapeutic effect in vivo. Overexpression of TIMP-3 resulted in apoptosis of A549 lung cancer cells and AdCMVTIMP3 up-regulated the expression of p53, Fas ligand, TNFR1 and TNFR2 on these cells. Adenoviral delivery of TIMP-3 gene inhibited the growth of pre-established A549 tumours in Balb/c nude mice, and was associated with a greater therapeutic effect than either TIMP-1 or -2 gene delivery. There was no evidence of increased hepatic toxicity following the delivery of TIMP-3 either from intra-tumoural or intravenous injection. Thus, at least in cells showing in vitro susceptibility, TIMP-3 gene therapy offers a therapeutic advantage over TIMPs 1 and 2. These findings establish the potential of adenoviral gene delivery of TIMP3 as a therapeutic agent for selected lung cancers.