The analysis of fibroin secretion-deficient 'naked-pupa' mutant silkworms has suggested that the disulfide linkage between heavy (H) and light (L) chains of fibroin, produced by the silkworm, Bombyx mori, is essential in its efficient large-scale secretion from the posterior silk gland cells. However, the site of disulfide-linkage between H- and L-chains has not been determined. In this study, cysteine residues involved in the single disulfide linkage between H- and L-chains were identified as the twentieth residue from the carboxyl terminus of H-chain (Cys-c20) and Cys-172 of L-chain by sequencing of genomic clones and peptide analysis. Furthermore, Cys-c4 (fourth residue from the carboxyl terminus) and Cys-c1 at the carboxyl terminus of H-chain were shown to form an intramolecular disulfide bond.