Abstract α 2-Antiplasmin (α 2AP) interferes with the binding of plasminogen to fibrin because lysine residues in its carboxy-terminal region compete with those in fibrin, presumably the same way that free lysine or ε-aminocaproic acid (EACA) inhibits plasminogen binding to fibrin. While this overall process causes an inhibition of fibrinolysis, the converse was observed with a 26-residue synthetic peptide (AP26) corresponding to the carboxy-terminal region of α 2AP. The AP26 peptide, in fact, accelerated urokinase-induced lysis of (1) fully crosslinked fibrin with complete γ-dimer and α-polymer formation; (2) partially crosslinked fibrin that had undergone only γ-dimerization; and (3) noncrosslinked fibrin. The AP26 peptide also inhibited factor XIIIa-catalyzed crosslinking of fibrin α-chains, and this also accelerated lysis of fibrin. EACA had no effect. In the presence of noncrosslinked fibrin, AP26 promoted plasminogen activation by urokinase and fibrinolysis. EACA only slightly increased the rate of plasminogen activation, and as expected, it inhibited fibrinolysis. Since AP26 peptide enhanced the lysis of partially crosslinked and noncrosslinked fibrin, our results indicate that inhibition of factor XIIIa-catalyzed α-polymer formation by AP26, although associated with accelerated fibrinolysis, is not the primary mechanism. Instead, our data support the conclusion that AP26 enhances the conversion of plasminogen to plasmin ∼5-fold, probably by inducing a conformational change in plasminogen structure just as occurs with low concentrations of lysine or EACA. At higher concentrations, however, AP26 apparently does not approach the avidity or affinity of lysine or EACA for the kringle structures of plasminogen or plasmin so that their binding to fibrin is blocked. Whether AP26 alone, or as part of another molecule, could have potential for enhancing thrombolysis will require further study.