Soybean Bowman-Birk proteinase inhibitor contains 14 half-cystine residues in a relatively short polypeptide chain of 71 amino acid residues, and five half-cystine residues around the trypsin-inhibitory site are in absolutely identical locations to those around the chymotrypsin inhibitory site. To assign the locations of the disulfide bridges, the native inhibitor was digested with a mold acid proteinase, thermolysin, pronase or subtilisin to yield cystine peptides suitable for structural investigation. These were separated and purified by chromatography on a Bio-Gel P-4 column and by paper electrophoresis. A peptide containing a -Cys-Cys- sequence was further hydrolysed with 10 N sulfuric acid. Amino acid analyses and appropriate amino terminal analyses of the resulting cystine peptides or their oxidized derivatives revealed the positions of 7 disulfide bridges in the parent molecule, which indicated the presence of two homologous trypsin- and chymotrypsin-inhibitory regions, and an almost symmetrical structure of the inhibitor.