Ancrod, a thrombin-like enzyme from Malayan pit viper venom, when infused into experimental animals or man converts fibrinogen into fibrin micro-clots. In a recent study we have found that in dogs pretreatment with ancrod markedly enhanced thrombolysis by recombinant tissue-type plasminogen activator (rt-PA), presumably by depleting fibrinogen and preventing new fibrin uptake by the thrombus during lysis. The rapid clearance of large amounts of fibrinogen from the circulation and the appearance of fibrin degradation products during ancrod treatment is indicative of high fibrinolytic activity. In this study we found that ancrod enhances activation of plasminogen by rt-PA indirectly via plasmin mediated digestion of fibrin. To a reaction mixture containing lys-plasminogen, human fibrinogen and val-leu-lys-pNA (S-2251), ancrod was added followed by rt-PA. At time intervals plasminogen activation was followed by measuring amidolytic activity on S-2251 at 405 nm. Ancrod or fibrinogen alone had no significant effect on the activation of plasminogen by rt-PA. However, the amidolytic activity increased with time in reaction mixture containing ancrod and fibrinogen. When a small amount of alpha-2-antiplasmin was added to the reaction mixture prior to the addition of rt-PA (the inhibitor level was sufficient to inhibit only a fraction of plasmin generated during the assay period) the activation rate was very much reduced. Thus, a small amount of plasmin initiates digestion of fibrin which may result in the exposure of new sites on fibrin that enhance the rate of activation of plasminogen by rt-PA. The rapid clearance of fibrinogen during ancrod infusion may not be due to increased susceptibility of micro-clots to lysis but to increased rate of plasmin formation. These results further confirm the observations of other investigators about the role of plasmin in the activation of plasminogen by tissue plasminogen activator.