From our established panel of monoclonal antibodies we have previously shown that an antibody binding to Domain 1 of uPAR, R3, completely inhibits the binding of DFP-inactivated '"I-uPA to U937 cells. In this assay another antibody, R5, which also reacts with Domain 1 of uPAR showed only a partial inhibition. The same inhibitory effects were seen in a DSS cross-linking assay using purified components. Using real-time biomolecular interaction analysis (BIA) it was possible to investigate the mechanisms involved in the antibody mediated inhibition of the uPA/uPAR interaction. The binding of R3 and uPA to uPAR are mutually exclusive, i.e competitive events. In contrast, a non-competitive inhibition was exerted by R5 in that this antibody actively dissociates preformed complexes between uPA and uPAR. Also, uPA disrupts preformed complexes of R5 and uPAR i.e the non-competitive inhibition is a mutually acting process. The apparent partial blocking of the uPA/uPAR interaction by R5 in cell-binding assay and cross-linking assay might then represent a partial disruption of the preformed uPAR/R5 complexes by uPA. Since the uPA/uPAR complex has a low dissociation rate, reagents that actively promote dissociation may represent an important alternative to competitive inhibitors in the attempts to develop uPAR antagonists for putative therapeutic use. The present study has shown that the structure of uPAR contains potential target areas for noncompetitive binding inhibitors, thus opening the possibility to pursue this strategy.