The half-cystine residues involved in linking the acidic and basic polypeptides were determined for several glycinin subunits. The cystines were localized with specific cyanogen bromide fragments either by comparing the electrophoretic mobility of nonreduced and reduced fragments, or by co-purifying and then determining the NH2-terminal sequence of the covalently linked fragments. Residues involved in the disulfides were further identified by labeling them with [3H] iodoacetic acid. Only 1 cystine was found to be involved in linking the acidic and basic components of each subunit, and they were in analogous positions in each of the subunits studied. Potential sites for intrapolypeptide cystines were also identified.