Thrombospondin-1 (TSP1) is a multifunctional matrix protein that influences the growth and function of a variety of normal and neoplastic epithelial and mesenchymal cell types. In vivo, TSP1 has shown potent antitumor activity in suppressing tumor neovascularization. Paradoxically, however, as we have reported, NIH 3T3 fibroblasts overexpressing TSP1 acquire the transformation-associated phenotypes of serum and anchorage independence in vitro but fail to form tumors in nude mice. To investigate these divergent results, and to determine the functional domains in TSP1 that confer serum and anchorage independence as well as antitumor and antiangiogenic activities, we transfected a series of deletion constructs of TSP1 into NIH 3T3 cells and into a v-src-transformed NIH 3T3 line. The antiangiogenic activity of TSP1-expressing, v-src-transformed NIH 3T3 cells was examined by assaying the conditioned media for inhibition of endothelial cell chemotaxis and suppression of basic fibroblast growth factor-mediated angiogenesis in the rat cornea. The link between TSP1 antitumor and antiangiogenic activities was assessed by measuring the rate of tumor growth and counting factor VIII-stained microvessels in the solid tumors developing in nude mice. Our results indicate that v-src NIH 3T3 cells transfected with a 449-amino acid N-terminal domain of TSP1 exhibit a dose-dependent suppression of tumor growth and neovascularization in nude mice. Truncated forms of TSP1 containing the type 1 properdin domain suppressed both endothelial cell chemotaxis and comeal neovascularization. Furthermore, when full-length TSP1 and deletion constructs containing the antiangiogenic type I properdin domain were transfected into highly tumorigenic v-src-transformed NIH 3T3 cells, they were able to confer transdominant suppression of tumorigenicity and angiogenesis of these cells in nude mice. These results confirm the role of TSP1 as a potent inhibitor of angiogenesis and provide support for the notion that alterations in the net balance between inducers and inhibitors of angiogenesis are largely responsible for the sustained growth of solid tumors in vivo.