Thrombospondin (TSP) forms specific complexes with transforming growth factor-β (TGF-β) in the α granule releasate of platelets and these TSP-TGF-β complexes inhibit the growth of bovine aortic endothelial cells (BAE). In these studies, we report that TSP stripped of associated TGF-β (sTSP) retained growth inhibitory activity which was partially reversed by a neutralizing antibody specific for TGF-β. Since BAE cells secrete latent TGF-β, we determined whether sTSP activates the latent TGF-β secreted by BAE cells. Cells were cultured with or without sTSP and then the conditioned medium was tested for the ability to support TGF-β-dependent normal rat kidney (NRK) colony formation in soft agar. Medium conditioned with sTSP showed a dose- and time-dependent ability to stimulate BAE-secreted TGF-β activity, reaching maximal activation by 1-2 h with 0.4 μg/ml (0.9 nM) sTSP. The sTSP-mediated stimulation of TGF-β activity is not dependent on serum factors and is not a general property of extracellular matrix molecules. The sTSP-mediated stimulation of TGF-β activity was blocked by a mAb specific for sTSP and by neutralizing antibodies to TGF-β. Activation of BAE cell secreted latent TGF-β by sTSP can occur in the absence of cells and apparently does not require interactions with cell surface molecules, since in conditioned medium removed from cells and then incubated with sTSP, activation occurs with kinetics and at levels similar to what is seen when sTSP is incubated in the presence of cells. Serine proteases such as plasmin are not involved in sTSP-mediated activation of TGF-β. Factors that regulate the conversion of latent to active TGF-β are keys to controlling TGF-β activity. These data suggest that TSP is a potent physiologic regulator of TGF-β activation.