Six murine monoclonal antibodies (MAbs) specific for urokinase-type plasminogen activator (u-PA) were tested for their ability to inhibit u-PA activity in three different assays with respect to amidolytic activity, plasminogen activation and fibrinolytic activity. Two of the MAbs were able to inhibit the amidolytic activity of u-PA. One MAb (LMW 3.3) inhibited at low concentrations, but only partially, whereas high concentrations of MAb LMW 2.3 could almost completely inhibit the u-PA activity. Testing the MAbs in the u-PA-mediated plasminogen activation assay, both in the presence and absence of fibrinogen fragments, and in the fibrinolytic assay, resulted in different inhibition patterns. The u-PA-mediated plasminogen activation in the presence of fibrinogen fragments was inhibited by high concentrations of all six MAbs. However, in the absence of fibrinogen fragments only MAb LMW 3.3 partially blocked the u-PA activity. In the u-PA-mediated fibrinolytic assay, besides MAb LMW 3.3, the kringle specific MAbs UK 2.1 and UK 50.2 were also able to inhibit the u-PA activity. The results indicate that the inhibition patterns of the MAbs can differ dramatically between different assays. In addition, the concentrations of the MAbs used in the assays can be very important as is illustrated by MAb UK 50.2, which showed in some assays a stimulating as well as an inhibitory effect dependent on the concentration of the MAb.