When blood plasma containing the NH2-terminal 12-residue peptide (N- peptide) of alpha 2-plasmin inhibitor (alpha 2PI; alpha 2-antiplasmin) was clotted in the presence of calcium ions, the N-peptide and alpha 2PI were cross-linked to fibrin by activated coagulation factor XIII. The amount of N-peptide cross-linked to fibrin was proportional to the concentration of N-peptide present in plasma. On the other hand, the amount of alpha 2PI cross-linked to fibrin was decreased by the presence of N-peptide, and the decrease was in reverse relationship to the increase of cross-linking of N-peptide. Spontaneous fibrinolysis or fibrinolysis induced by tissue plasminogen activator was accelerated by the presence of N-peptide, and the acceleration was dependent on the concentrations of N-peptide and directly proportional to inhibition of alpha 2PI cross-linking exerted by N-peptide. The acceleration was more pronounced when the clot was compacted by platelet-mediated clot retraction or by a squeeze. Fibrinolysis of an alpha 2PI-deficient or a factor XIII-deficient plasma clot was not accelerated by N-peptide. These findings were substantiated in a purified system and support the previous proposal that alpha 2PI is cross-linked to fibrin at the glutamine residue that is next to the NH2-terminus of alpha 2PI, and this factor XIII-mediated cross-linking of alpha 2PI is significant in inhibition of physiologically occurring endogenous fibrinolysis.