Transforming growth factor-β (TGF-β) is secreted as a latent complex of the latency-associated peptide (LAP) and the mature domain, which must be activated for TGF-β to signal. We previously identified thrombospondin 1 (TSP1) as a physiologic activator of TGF-β in vitro and in vivo. The WSXW sequences in the type 1 repeats of TSP1 interact with the mature domain of TGF-β, and WSXW peptides inhibit TSP1-mediated activation by blocking TSP1 binding to the TGF-β latent complex. However, the binding site for the WSXW sequence was not identified. In this report, we show that the WSXW sequences bind the 61VLAL sequence in mature TGF-β and also bind 77VLAL in LAP. A glutathione S-transferase (GST) fusion protein of the second TSP1 type 1 repeat (GST-TSR2) binds immobilized VLAL peptide. VLAL peptides inhibit binding of LAP and mature TGF-β to soluble GST-TSR2 and immobilized WSXW peptide. VLAL peptide inhibits TSP1-mediated activation of recombinant and endothelial cell-derived latent TGF-β. Furthermore, TGF-β or LAP deleted in the VLAL sequence fails to bind immobilized WSXW or soluble GST-TSR2, indicating that binding to both VLAL sequences is important for association of TSP1 and the latent complex. Additionally, TSP1 is unable to activate latent TGF-β when VLAL is deleted from the mature domain. These data show that the WSXW motif binds VLAL on both LAP and mature TGF-β, and these interactions are critical for TSP1-mediated activation of the TGF-β latent complex.