A cross-linked polypeptide chain involved in stabilized bovine fibrin was isolated by two cycles of gel-filtration on a Sepharose 6B column and chromatography on a CM-cellulose column after sulfitolysis of the parent protein. The isolated chain designated as “X2” was homogeneous electrophoretically. The molecular weight of “X2” was estimated to be approximately 110, 000 by the method of Andrews. This value is significantly different from the average molecular weight of 58, 000 of the α, β and γ-chain peptides in fibrin. However, the amino acid compositions, N-terminal residues and peptide mappings of “X2” and the Γ-chain peptide were quite similar. The N-terminal residue of both was tyrosine, and their tryptic peptide maps differed only in the distributions of a few spots. Furthermore, no tryptic peptides derived from the α- and β-chain peptides were detected in the peptide map of “X2,” suggesting that the α- and β-chains were not involved in the cross-links. The following conclusion are drawn from these results: (1) The cross-linked chain, “X2” isolated from S-sulfo-stabilized fibrin is composed of the Γ-chain peptide, one of the three subunits of the fibrin molecule. “X2” seems to contain two Γ-chain peptides. (2) The chain peptides which are linked together must contain both donor and acceptor sites respectively, to construct the cross-linkage, ε-(Γ-glutamyl)-lysine. (3) Thus, it seems that the cross-linkage, which stabilizes the fibrin clot formed by the action of activated FSF, mainly involves a pair of Γ-chain peptides in the fibrin molecule.