Tissue plasminogen activator (t-PA) and/or pro-urokinase (pro-UK) induced lysis of standard 125I-fibrin clots suspended in plasma was studied. Doses were kept below the concentration at which a nonspecific effect was seen, i.e., where fibrinogenolysis and major plasminogen consumption were observed. Small amounts of t-PA potentiated clot lysis by pro-UK by attenuating the lag phase characteristic of pro-UK, and causing a much earlier transition to the rapid phase of lysis. Similar promotion of the fibrinolytic effect of pro-UK was obtained when clots were pretreated with UK or with a little plasmin (less than 1% clot lysis). Promotion by plasmin was nullified by a subsequent treatment of the clot with carboxypeptidase B, indicating that the plasmin effect was related to the exposure of carboxy terminal lysine residues on fibrin. These lysine termini, absent in undegraded fibrin, are known to be essential for the high affinity binding of plasminogen to fibrin. In contrast, clot lysis by t-PA was unaffected by plasmin pretreatment and little affected by carboxypeptidase B treatment of the fibrin substrate. Therefore, plasminogen bound to lysine termini on fibrin, although found to be essential for pro-UK, did not appear to serve as a substrate for t-PA. Selective activation of fibrin bound plasminogen has been attributed to the conformational change in Glu-plasminogen that occurs as a result of binding. The present findings suggest that this conformational change occurs when plasminogen is bound to a terminal lysine but not to an internal lysine. Plasminogen bound to the latter site on fibrin was activated by t-PA and therefore is involved in the ternary complex. This initiates lysis of the undegraded clot and exposes the plasminogen binding sites required by pro-UK. By their complementary activation of fibrin bound plasminogen, t-PA followed by pro-UK induces efficient and synergistic fibrinolysis, whereas each is relatively inefficient when used alone.