Formation of insulin-glutathione mixed disulfides takes place under the conditions of 0.1 M ammonium acetate, neutral pH and without the presence of any enzyme. Using a SH-free glutathione-agarose column it is demonstrated that the interaction of insulin with glutathione is specific, and increasing the incubation time between these two peptides results in the reduction of insulin disulfide bonds and the production of A and B chains.