Protein-disulfide reductase (glutathione-insulin transhydrogenase, EC 1.8.4.2) was isolated from bovine liver and purified by chromatography on Sephadex G-100, with a relatively high yield and a maximal specific activity of 6000 units (as defined by Tomizawa and Halsey). The enzyme was homogeneous in electrophore-sis; flavin groups could not be detected. The molecular weight was about 200,000, calculated from gel filtration on Sephadex G-200. The cleavage of insulin by SH-compounds in the presence of the transhydrogenase (and the slight cleavage in the absence of enzyme) depends on the redox potential of the compound, and is otherwise independent of its chemical constitution. The chemically reduced A- and B-chains of insulin are reoxidized to give insulin activity under the influence of the transhydrogenase and disulfides of various potentials. The extent of this reoxidation depends on the redox potential and, above all, on the concentration of the disulfide. Insulin activity was determined in the rabbit, in the epididymal fatpad of the rat and partly by the immunolo-gical insulin test. The enzyme probably lowers the activation energy of the disulfide exchange, since its SH-groups form mixed disulfides alternately with the insulin chains and the thiol reagents, during both the resynthesis and the cleavage reactions. No experimental evidence could be obtained for the participation of the enzyme in the air-auto-oxidation process.