We have demonstrated previously that overexpression of tissue inhibitor of metalloproteinases-2 (TIMP-2), an inhibitor of matrix-degrading metalloproteinases, not only inhibits the invasive and metastatic behavior of tumor cells but also significantly decreases tumor growth in vivo (Y. A. DeClerck et at, Cancer Res., 52: 701-708, 1992). This latter effect was found to be dependent on the ability of TIMP-2 to prevent the degradation of the collagen matrix (A. M. Montgomery et al., Cancer Res., 54: 5467-5473, 1994). In this report, we have overexpressed TIMP-2 in tumor tissue by retroviral-mediated gene transfer into tumor cells by co-injecting s.c. in nude mice tumorigenic c-Ha-ras-transfected rat embryo fibroblasts with irradiated packaging cells producing high titer retroviral vectors containing the human TIMP-2 cDNA. The growth rate of tumors derived from cells co-injected with the TIMP-2 vector producer cells was significantly slower than the growth rate of tumors derived from cells co-injected with packaging cells producing a retrovirus containing the Escherichia coli beta-galactosidase gene. The transduction efficiency was estimated at 13%, and the production of a functional human TIMP-2 in tumor cells transduced with the TIMP-2-containing vector was documented. Furthermore, histological analysis of tumors derived from tumor cells co-injected with the TIMP-2 vector producer cells revealed the presence of a thick connective tissue capsule and a lack of local invasion. The data indicate that retroviral-mediated transduction of TIMP-2 cDNA into a limited population of tumor cells in vivo is sufficient to increase the accumulation of connective tissue proteins in tumor tissue, to inhibit growth, and to prevent local invasion.