Thrombospondin (TSP) is complexed with transforming growth factor-β (TGF- β) in the α-granules of stimulated platelets. TSP stripped of associated TGF-β activity (sTSP) activates latent TGF-β secreted by bovine aortic endothelial cells (BAE) in culture. To better understand the interactions of TSP with TGF-β, we investigated which region of sTSP interacts with TGF-β. The chymotrypsin-resistant core of TSP, which contains the procollagen-like region and the properdin-like type 1 repeats, activated both latent TGF-β secreted by BAE and a recombinant form of the small latent TGF-β complex at levels similar to or better than sTSP. The core fragment bound 125I-TGF- β in solution and shifted the elution profile of 125I-TGF-β in gel permeation chromatography. Fusion constructs of the type 1, 2, and 3 repeats and the COOH terminus of TSP1 were tested for their ability to activate latent TGF-β. Only the type 1 construct, containing the three properdin- like repeats of TSP found in the 50-kDa fragment, activated latent TGF-β. In addition, a polyclonal antibody against the type 1 construct inhibits activation of latent TGF-β by intact TSP, suggesting that this region is exposed in the intact molecule. These results show that the type 1 properdin- like repeats of TSP are responsible for activating recombinant and endothelial cell-derived latent TGF-β and that this site is exposed in intact TSP.