Two plasma proteins, alpha 2-plasmin inhibitor and plasma fibronectin, are cross-linked to fibrin by plasma transglutaminase (R-glutaminyl-peptide : amine gamma-glutamyl-yltransferase, EC 220.127.116.11, fibrin stabilizing factor) when blood coagulation takes place. The cross-linking reactions of these proteins were analyzed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS) using these radioactively labeled proteins. Both proteins were cross-linked exclusively to the alpha-chain of fibrin, and each of these cross-linking reactions proceeded independently without being influenced by the other cross-linking reaction. The cross-linking of fibronectin to the alpha-chain proceeded steadily at a rate similar to that of the cross-linked polymerization of the alpha-chain. In contrast, the cross-linking reaction of alpha2-plasmin inhibitor to fibrin proceeded markedly faster than that of fibrin polymerization but did not proceed further after reaching a certain relatively low level of cross-linking. Most of the cross-linked alpha 2-plasmin inhibitor molecules at this stage of the fibrin cross-linking were in the form of complex with the alpha-chain monomer. The complex with the alpha-chain monomer was gradually transformed to a complex with the alpha-chain polymer as the cross-linking polymerization of the alpha chain proceeded. The rate of the transformation was the same as that for the disappearance of the alpha-chain monomer, indicating that whether the alpha-chain was cross-linked to alpha 2-plasmin inhibitor or not, the alpha-chain underwent cross-linking polymerization at the same rate.