SP-40,40, a human plasma protein, is a modulator of the membrane attack complex formation of the complement system as well as a subcomponent of high-density lipoproteins. In the present study, the positions of the disulfide bonds in SP-40,40 were determined. SP-40,40 was purified from human seminal plasma by affinity chromatography using an anti-SP-40,40 monoclonal antibody and reversed-phase, high-performance liquid chromatography (HPLC). The protein was digested with trypsin and the fragments were separated by reversed-phase HPLC. The peptides containing disulfide bonds were fluorophotometrically detected with 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (ABD-F). The peptides containing more than two disulfide bonds were further digested with Staphylococcus aureus V8 protease and lysylendopeptidase, and the fragments were isolated by HPLC. The amino acid compositions and the amino acid sequences of the peptides containing only a disulfide bond were determined. Disulfide bonds thus determined were between Cys58(alpha)-Cys107(beta), Cys68(alpha)-Cys99(beta), Cys75(alpha)-Cys94(beta), and Cys86(alpha)-Cys80(beta). Since there was no free sulfhydryl groups in the SP-40,40 molecule, Cys78(alpha) and Cys91(beta) should also be linked by a disulfide bond. It is notable that all of the disulfide bonds in SP-40,40 are not only formed by inter-chain pairing, but also appear to form an antiparallel ladder-like structure between the two chains. The unique structure could be related to the functions of SP-40,40.