32P-labeled perfused rat hearts were used to study the hormonal regulation of glycogen synthase. Following equilibration of perfused hearts with inorganic [32P]phosphate for 30 min, there was an incorporation of approximately 200 pmol of [32P]phosphate/unit of enzyme activity that arose from an exchange of [32P] phosphate with the endogenous [31P]phosphate. Maximum insulin-induced activation (10 milliunits/ml for 5 min), which promoted an I/D activity ratio change from 25% I to 40% I, was associated with a 22% decrease in phosphate content of the enzyme. With hearts from alloxan-induced diabetic animals, there was a 17% higher level of phosphate incorporation and a 4-fold decrease in % I glycogen synthase activity compared to normal animals, but with the diabetic tissue, insulin added to the perfusate had no effect on either the phosphate content or the activity ratio of the enzyme. In perfused hearts from normal animals, DL-isoproterenol and glucagon caused an increase in glycogen synthase phosphorylation of 85-100 pmol/unit of enzyme activity, while L-phenylephrine increased the phosphate content by only 20-35 pmol, but all three hormones caused the same degree of inactivation. The increase in cardiac glycogen synthase phosphorylation induced by DL-isoproterenol, glucagon, and L-phenylephrine was identical, with or without insulin pretreatment; this despite the fact that these three hormones promoted a 3- to 4-fold larger decrease in the enzyme activity ratio with the insulin-treated tissue. In perfused diabetic hearts, DL-isoproterenol, glucagon, and L-phenylephrine caused increased phosphorylation of glycogen synthase without affecting the albeit already low activity ratio of the enzyme. These results show that in the intact perfused heart the same degree of glycogen synthase inactivation can occur as a consequence of differing degrees of phosphorylation, presumably due to phosphorylation at different sites promoted by different second messengers. Conversely, the data indicate that the same extent of phosphorylation, as stimulated by the same second messenger, can inactivate glycogen synthase by different amounts depending upon the prior phosphorylation state of other sites in the protein.