Tat protein, a trans-activating factor of the human immunodeficiency virus type 1, acts also as an extracellular molecule modulating gene expression, cell survival, growth, transformation, and angiogenesis. Here we demonstrate that human thrombospondin-1 (TSP), a plasma glycoprotein and constituent of the extracellular matrix, binds to glutathione-S-transferase (GST)-Tat protein but not to GST. Scatchard plot analysis of the binding of free GST-Tat to immobilized TSP reveals a high-affinity interaction (Kd equal to 25 nM). Accordingly, TSP inhibits cell internalization and HIV-1 LTR trans-activating activity of extracellular Tat in HL3T1 cells with ID50 equal to 10-30 nM. Also, TSP inhibits cell interaction and mitogenic activity of extracellular Tat in T53 Tat-less cells. TSP is instead ineffective when administered after the interaction of Tat with cell surface heparan-sulfate proteoglycans has occurred, in keeping with its ability to prevent but not disrupt Tat/heparin interaction in vitro. Finally, TSP inhibits the autocrine loop of stimulation exerted by endogenous Tat in parental T53 cells. Accordingly, TSP overexpression inhibits cell proliferation, angiogenic activity, and tumorigenic capacity of stable T53 transfectants. Our data demonstrate the ability of TSP to bind to Tat protein and to affect its LTR trans-activating, mitogenic, angiogenic, and tumorigenic activity. These findings suggest that TSP may be implicated in the progression of AIDS and in AIDS-associated pathologies by modulating the bioavailability and biological activity of extracellular Tat.