Transforming growth factor-β (TGF-β) is a potent growth regulatory protein normally secreted by cells in a latent form. Primary regulation of TGF-β activity occurs through factors which control the processing of the latent to the biologically active molecule. Thrombospondin (TSP), a platelet α-granule and extracellular matrix protein, forms specific complexes with active TGF-β in platelet releasate and activates endogenous latent TGF-β secreted by endothelial cells via a cell- and protease-independent mechanism. In order to better understand TSP-mediated activation of cell-secreted latent TGF-β, we examined the consequences of interactions of the large (platelet- derived) and small (recombinant) forms of latent TGF-β with TSP in a chemically defined system. Data from these studies show that interactions between TSP and both forms of latent TGF-β result in the generation of biologically active TGF-β as assayed by the ability of NRK-49F cells to form colonies in soft agar, by the ability to compete for binding to TGF-β receptors on endothelial cells, and by an enzyme-linked immunosorbent assay selective for the active form of TGF-β. Activation of latent TGF-β by TSP stripped of associated TGF-β activity (sTSP) is time- and concentration- dependent, but temperature-independent. The mechanism whereby sTSP activates latent TGF-β appears to involve the direct binding of sTSP to the latent molecule as shown by gel permeation chromatography. In addition, a polyclonal antibody specific for the amino-terminal region of the latency-associated peptide (amino acids 81-94) inhibits sTSP-mediated activation of latent TGF- β in both the chemically defined system and in endothelial cell conditioned medium. These data and the observation that similar concentrations of sTSP activate latent TGF-β in both the chemically defined system and in the endothelial cell system indicate that there is a common mechanism by which TSP activates the small, large, and endothelial cell-derived latent TGF-β complexes. The ability of TSP to convert latent TGF-β to biologically active TGF-β suggests that TSP is a major regulatory factor in the control of TGF- β activity.