Stanniocalcin (STC) is a hypocalcemic glycoprotein hormone secreted by the corpuscles of Stannius, endocrine glands unique to the bony fishes. Recently, two cDNAs encoding proteins, STC-1 and STC-2, homologous to fish STC have been identified in humans and rodents. The STC-1, but not STC-2, is identical to fish STC with respect to location of all Cys residues. The present study reports the localization of inter- and intra-molecular disulfide linkages in chum salmon STC. The chum salmon STC was deglycosylated and digested with several proteases in series. Six fragments, each of which consisted of two peptides connected by a single disulfide bond, were isolated by HPLC. Disulfide bonds were determined by sequence analysis after reduction and S-alkylation of each peptide fragment. Chum salmon STC is a homodimer connected by a single intermonomeric disulfide bond at Cys169. The monomer consists of 179 amino-acids, containing five intramonomeric disulfide bonds formed between Cys12-Cys26, Cys21-Cys41, Cys32-Cys81, Cys65-Cys95, and Cys102-Cys137.