An ELISA has been developed for the assessment of complexes between the urokinase-type (uPA) and the tissuetype plasminogen (tPA) activators with their inhibitor type-1 (PAI-1) in cell-culture medium and cytosolic extracts of breast tumours. The ''4-stage/2-site'' ELISA involves 2 polyclonal antibodies in the pre-analyte stage 2 and in the post-analyte stage. For the specific measurement of the uPADPAI-1 complex, 2 assay formats may be employed, uPA/PAI-1 and PAI-1/uPA. This offers an attractive facility for quality-assessment studies of this kind of assays. Analogously, the tPADPAI-1 complex was assessed using the formats tPA/PAI-1 and PAI-1/tPA. Only complexes are able to evoke a signal in their appropriate assay formats. The free component, however, which responds to the capture antibody, could interfere with the binding of the complex molecule, reducing the OD signal. Increasing the coating Ab concentration diminishes the signal-suppressing effect of the free component. In 15 cell-culture supernatants, uPA and PAI-1 concentrations were measured as well as the uPA of PAI-I complex in different dilutions in 2 assay formats. The differences between the values of complex measured in the 2 assay formats could be accounted for by the free uPA and PAI-1 concentrations. At dilution 1:10, the measured values obtained in the 2 separate formats differed substantially (correlation coefficient r ؍ 0.641). At dilution 1:20, the differences were already smaller between the values (agreement 0.945). At dilution 1:30, close agreement between the corresponding values was observed (r ؍ 0.971). Extrapolation to infinite dilution of the results obtained resulted in an even closer estimation of the complex concentration. Comparable results have been observed when tPA, PAI-1 and tPADPAI-1 values were measured in tumour biopsy extracts.