Different extracellular matrix proteins have been described as binding proteins for growth factors, influencing their storage or presentation towards cellular receptors. The multifunctional adhesive glycoprotein vitronectin (VN), which is found in the circulation and widely distributed throughout different tissues, has been implicated in the regulation of vascular cell functions, and these activities could be related to interactions with various growth factors. In vitro, soluble VN interfered with transforming growth factor-β (TGF-β) binding to isolated extracellular matrix and was found to associate with TGF-β1 and TGF-β2 as well as with other growth factors such as vascular endothelial growth factor, epidermal growth factor, or basic fibroblast growth factor in a saturable manner. In particular, binding of TGF-β was maximal for the heparin-binding multimeric isoform of VN, whereas VN in a ternary complex with thrombin and antithrombin or plasma VN exhibited weaker binding. Plasminogen activator inhibitor-1 (PAI-1) or heparin, but not desulfated glycosaminoglycans, interfered with binding of VN to TGF-β, and soluble PAI-1 was able to dissociate VN-bound TGF-β. Upon limited plasmin proteolysis of VN, only the fragments comprising the intact aminoterminal portion of VN bound to TGF-β as did a synthetic peptide (amino acids 43 to 62), indicating that TGF-β and PAI-1 share common binding site(s) on VN. Although VN did not influence TGF-β bioactivity for mink lung epithelial cells, TGF-β dose dependently inhibited both urokinase-receptor as well as α_v-integrin-dependent adhesion to VN. This activity of TGF-β was reminiscent of the antiadhesive function of PAI-1. In atherosclerotic tissue sections, staining patterns of VN and TGF-β indicated their colocalization. These findings describe VN as a new binding protein for TGF-β, whereby specific functions of both factors become modulated by this interaction.