Rat liver glycogen synthetase D is strongly inactivated by a variety of natural disulfides but not by the corresponding sulfhydryl compounds. Oxidized glutathione was used as a model compound in studies on the mechanism of inactivation. Inactivation by oxidized glutathione (GSSG) is a time- and concentration-dependent process and results in the formation of an inactive, stable enzyme species. Inactivated enzyme can be reactivated by incubation in the presence of various sulfhydryl compounds including glutathione (GSH). The extent of inactivation and reactivation depends upon the presence of glucose-6-P during the two processes. Binding studies with [³⁵S]GSSG revealed simultaneous incorporation of ³⁵S into glycogen synthetase D and loss of enzyme activity. Reactivation with GSH was accompanied by the release of ³⁵S label from the enzyme. Inactivation of glycogen synthetase is the result of the formation of mixed disulfides between GSSG and the sulfhydryl group(s) of glycogen synthetase D.