The chemical reactivity of disulfide bonds towards reducing agents, in the absence of denaturing conditions, in cholera toxin has been studied. Treatment of the toxin with dithiothreitol or other mercaptans gave selective reduction of one of the six disulfide bonds of the protein. This reactive disulfide links two distinct functional regions of the toxin, fragment alpha, which activates adenylate cyclase, and fragment gammabeta5, which recognizes the cell surface receptors. Upon reduction, the two fragments remain bound together and the secondary structure of the protein is retained. The two functional regions have been separated and purified only by methods based on charge differences. When mixed together, purified alpha and purified gammabeta5 fragments spontaneously and rapidly re-form the disulfide bond. However, reduction of the disulfide bond is an absolute requirement for freeing the catalytic site of the alpha functional region. Thus, while other non-covalent binding regions are involved in maintaining cholera toxin molecular structure, the reactive disulfide bond may play a role in the mechanism of cell intoxication.