The urokinase receptor (uPAR) is a high affinity receptor for both urokinase plasminogen activator (uPA) and the adhesion protein vitronectin and is a central component in pericellular proteolysis and cellular adhesion. Two forms of cell surface bound uPAR are present on many cell types; the lull length uPAR and a cleaved form, uPAR(2+3). On cultured U937 cells uPAR(2+3) is formed by uPA catalyzed cleavage of uPAR. In ligand-blotting experiments using uPAR purified from U937 cell lysates we found, that vitronectin and uPA bind uPAR but not uPAR(2+3). In order to study various aspects of vitronectin binding to intact and cleaved forms of uPAR we employed BIA technology and recombinant, soluble uPAR (suPAR) preparations. Both plasma and multimeric forms of vitronectin bound to intact, antibody-immobilized suPAR and uPA augmented this binding. Plasminogen activator inhibitor 1 (PAI-1) inhibited suPAR binding to vitronectin in a dose-dependent manner with maxima] inhibition at equimolar concentrations of vitronectin and PAI-1. Monoclonal antibodies against domain 1 of uPAR blocked suPAR binding to vitronectin and vitronectin did not interact with suPAR(2+3). Both suPAR(2+3) and the isolated domain 1 failed to compete with the intact suPAR in binding to vitronectin, indicating that the intact receptor is required for high affinity vitronectin binding. Thus, cleavage of uPAR prevents cell surface potentiation of plasminogen activation as well as uPAR-dependent vitronectin binding with consequences for cellular adhesion .