Jararhagin, a haemorrhagic metalloproteinase from Bothrops jararaca venom, plays an important role in systemic as well as local haemorrhage. In this study, the effect of jararhagin on the fibrinolytic system was investigated. The fibrinolytic activity of various kinds of animal plasmas was measured by the fibrin plate method. No activity was detected in plasma alone. However, after mixing plasma with jararhagin, strong fibrinolytic activity was recorded in guinea-pig, horse, dog, rabbit and human plasmas. The mechanism of the increase of fibrinolytic activity by jararhagin was studied further in guinea-pig plasma. Fibrin-zymographic studies indicated that jararhagin increased tissue-type plasminogen activator (tPA) activity by the dissociation of a complex of tPA with type 1 plasminogen activator inhibitor (PAI-1). α 2-Plasmin inhibitor ( α 2-PI) activity in the plasma was measured using a synthetic chromogenic substrate method after incubation with jararhagin. The α 2-PI activity in the plasma decreased in both time-dependent and dose-dependent manners. These in vitro results suggest that, in some animal plasmas, jararhagin increases plasma fibrinolytic activity by causing dissociation of the tPA/PAI-1 complex and by the inactivation of α 2-PI. It is possible that this direct action of jararhagin on the enhancement of plasma fibrinolytic activity may contribute to the aetiology of systemic haemorrhage frequently observed in human victims of B. jararaca envenoming.