During blood coagulation alpha 2-plasmin inhibitor (alpha 2PI) is cross-linked with fibrin by an activated fibrin-stabilizing factor (FSFa) plasma transglutaminase, activated coagulation factor XIII). When alpha 2PI was treated with FSFa in the absence of acceptor amino groups, the inhibitor lost more than 90% of its capacity to be cross-linked to fibrin because of hydrolysis of the gamma-carboxamides of FSFa-susceptible glutamine residues. Chemical modifications of the inhibitor's lysine epsilon-amino groups did not affect the cross-linking capacity of the inhibitor with fibrin, whereas the same chemical modifications in fibrinogen resulted in a remarkable loss of cross-linking capacity. These observations suggest that alpha 2PI plays a role as an acyl donor with its FSFa-susceptible glutamine residues in the cross-linking reaction with fibrin, and fibrin serves as an acyl acceptor with its lysine residues. The number of FSFa-susceptible glutamine residues/molecule of the inhibitor was estimated by measuring the maximum incorporation of [3H]histamine into the inhibitor and by analyzing the distribution of radioactivity in a tryptic digest of [14C]histamine-incorporated alpha 2PI.l It was found that each inhibitor molecule has one glutamine residue that is most susceptible to FSFa. When the radioactive histamine-incorporated inhibitor was reacted with excess amounts of plasmin, a small fragment carrying all the released radioactivity was rapidly released from the NH2-terminal part of the inhibitor moiety of the complex. The NH2-terminal amino acid sequence of the inhibitor was analyzed before and after treatment with FSFa or before and after incorporation of radioactive histamine. The glutamine residue at the second position from the NH2-terminal end was converted to a glutamic acid residue when the inhibitor was treated with FSFa. When the radioactive histamine-incorporated inhibitor ws analyzed, the radioactivity was found predominantly at the second position from the NH2-terminal end. These results indicate that the glutamine residue susceptible to FSFa in alpha 2PI is located next to the NH2-terminal residue.