The effect of fibrin-targeting of urokinase-type plasminogen activator (u-PA) on its fibrinolytic potency was studied using recombinant fusion proteins of u-PA with the NH2-terminal region of tissue-type plasminogen activator (t-PA/u-PA) and chemical complexes of u-PA with F(ab')2 fragments of a fibrin specific monoclonal antibody (u-PA/MA-15C5-F(ab')2). Two chain derivatives of a low Mr variant of u-PA comprising amino acids Leu144-Leu411 (tcu-PA-32k), obtained by cleavage of recombinant single-chain u-PA (rscu-PA-32k) with thrombin (rtcu-PA-32k/T) or plasmin (rtcu-PA-32k/P) were investigated. The plasmin-derived two chain u-PA moieties, rtcu-PA-32k/P, rt-PA/tcu-PA-32k/P and rtcu-PA-32k/MA-15C5-F(ab')2/P had high specific activities in amidolytic and fibrin plate assays (130,000 and 150,000 IU/mg u-PA, 43,000 and 71,000 IU/mg u-PA and 32,000 and 56,000 IU/mg u-PA respectively). The thrombin-derived two chain u-PA moieties had a very low amidolytic activity, corresponding to less than or equal to 1 percent of that of their plasmin-derived counterparts. On fibrin plates, however, rtcu-PA-32k/T had a negligible activity, whereas rt-PA/tcu-PA-32k/T and rtcu-PA-32k/MA-15C5-F(ab')2/T had specific activities of 12,000 and 25,000 IU/mg u-PA respectively. The catalytic efficiency for plasminogen activation of rtcu-PA-32k/MA-15C5-F(ab')2/T is 4,000-fold lower than that of rtcu-PA-32k/MA-15C5-F(ab')2/P, but its concentration required for 50 percent lysis in 2 hours of a 125I-fibrin labeled plasma clot in human plasma (C50) is only 25-fold higher. The catalytic efficiency of rt-PA/tcu-PA-32k/T is 1,600-fold lower and the C50 100-fold higher than that of rt-PA/tcu-PA-32k/P. The catalytic efficiency and the fibrinolytic potential of rtcu-PA-32k/T are negligible as compared to that of rtcu-PA-32k/P. These observations may be explained by conversion of the thrombin derived two chain u-PA moieties to their plasmin-derived analogues at the fibrin surface. This conversion appears to be most efficient for the antibody conjugate which has a high fibrin-affinity, less efficient for the t-PA/u-PA chimera which has only moderate fibrin-affinity, and negligible for the unconjugated u-PA moiety which has no fibrin-affinity. These findings illustrate the importance of plasmin-mediated positive feedback mechanisms in u-PA mediated clot lysis.