We compared the fibrinolytic properties of recombinant staphylokinase (SAK), a fibrin-specific plasminogen activator, with those of streptokinase and tissue-type plasminogen activator (t-PA) by means of the amidolytic method. We also investigated the involvement of α₂-macroglobulin, C₁-inactivator and α₁-antitrypsin in SAK-induced fibrin-specific fibrinolysis. Both SAK and t-PA activated plasminogen efficiently in the presence of fibrin in human plasma. Although t-PA activated plasminogen dependently on fibrin in the reconstituted plasma system, SAK activated plasminogen independently of fibrin without α₂-plasmin inhibitor (α₂-antiplasmin, α₂-PI). These findings suggest that fibrin and α₂-PI play important roles in plasminogen activation by SAK but not by t-PA. Furthermore, protease inhibitors such as α₂-PI, α₂-macroglobulin, C₁-inactivator and α₁-antitrypsin inhibited plasminogen activation by SAK and the inhibitory actions of these protease inhibitors disappeared in the presence of fibrin. This shows that α₂-macroglobulin, C₁-inactivator and α₁-antitrypsin, other than α₂-PI, contribute to the fibrin-specificity of SAK.