The urinary protein C inhibitor (PCI) binding region in the Bβ-chain of human fibrinogen was examined by ligand blotting, reversephase HPLC and amino acid sequencing. The Bβ-chain, isolated from reduced and pyridylethylated fibrinogen, was digested with staphylococcal V8 protease to yield eight peptides consisting of 10, 12, 13, 13.5, 14, 16, 17 and 18 kDa bands and the cleaved peptides were ligand-blotted. The 12 kDa band bound to urinary PCI. Moreover, the digested Bβ-chain was isolated by reverse-HPLC and the elution peak showing positive binding to urinary PCI was sequenced. The N-terminal amino acid sequence was A V S Q T, which corresponds to Ala106- Thr110 in the Bβ-chain. Judging from theM,of this peptide (12 kDa), it comprises the region from Ala106 to Glu192-Glu210 which are partly located in the D-region. It is concluded that the urinary PCI-binding region to the Bβ-chain resides in the sequence of Ala110 to Glu192-Glu210.