The mechanism of the activation of plasminogen by recombinant pro-urokinase (Rec-pro-UK), obtained by expression of the human pro-urokinase gene in Escherichia coli, was investigated in purified systems. In mixtures of Rec-pro-UK and plasminogen, both active urokinase and plasmin are quickly generated. Addition of plasmin inhibitors (aprotinin or alpha 2-antiplasmin) abolishes the conversion of Rec-pro-UK to urokinase but not the activation of plasminogen to plasmin, suggesting that Rec-pro-UK activates plasminogen directly. Human plasma competitively inhibits the activation of plasminogen by pro-urokinase with a Ki of 0.2% (v/v). This explains the relative stability of Rec-pro-UK in plasma and the lack of activation of the plasma fibrinolytic system in the absence of fibrin. The competitive inhibition by plasma is abolished by the addition of CNBr-digested fibrinogen although Rec-pro-UK has no specific affinity for fibrin. These findings suggest that the fibrin specificity of the activation of plasminogen by pro-urokinase is due to neutralization by fibrin of the competitive inhibition exerted by plasma and not to fibrin-enhanced activation of plasminogen.