The in vivo phosphorylation state of glycogen synthase was re-examined by fast-atom-bombardment mass spectrometry and a procedure in which phosphoserine residues are first converted to S-ethylcysteine. In animals injected with the /3-adrenergic antagonist propranolol, the phosphorylation sites in the N-terminal (N) and Cterminal (C) cyanogen bromide peptides were identified as the serine residues at N7, the region C28 -C39, C42, C46 and C100. In animals injected with adrenalin, the phosphorylation of N7 increased from 0.6 to 0.8 mol/mol, the region C28 -C39 from 0.7 to 1.2 mol/mol and ClOO from 0.3 to 0.6 mol/mol. The phosphorylation states of C42 (0.7 mol/mol) and C46 (0.9 mol/mol) were unchanged. In addition, two further serine residues became phosphorylated at positions N10 (0.5 mol/mol) and C87 (0.5 mol/mol), which were not phosphorylated in the absence of adrenalin.Residues N10 and C42 have not been recognized as in vivo sites of phosphorylation previously. The results suggest that N10 is phosphorylated by a novel protein kinase which may be activated by cyclic-AMP-dependent protein kinase. The phosphorylation of C42 is likely to be catalysed by glycogen synthase kinase 3. The protein kinases responsible for phosphorylating N7, the region C28 -C39, C46, C87 and ClOO in vivo and the molecular mechanisms by which adrenalin inactivates glycogen synthase in vivo are discussed.Residue N3, a major site phosphorylated by casein kinase-I in vitro is not phosphorylated in vivo. This and other evidence indicates that casein kinase-I is not a glycogen synthase kinase in vivo.Glycogen synthase, the rate-limiting enzyme in glycogen synthesis, is regulated by multisite phosphorylation. Up to 1983, seven phosphorylation sites had been identified in the rabbit skeletal muscle enzyme, all located in the amino-terminal (N) or carboxy-terminal (C) cyanogen bromide peptides [l, 21. The amino acid sequences of these regions are shown in Fig. 1. In the N-terminal domain, residue N7 was phosphorylated in vitro by the calmodulin-dependent multiprotein kinase [3 -51, phosphorylase kinase [6,7], cyclic-AMP-dependent protein kinase [8] and glycogen synthase kinase-4 [9]. In the C-terminal domain, glycogen synthase kinase-3 phosphorylated three residues C30, C34 and C38, all contained within the same tryptic peptide [lo], while casein kinase-11 labelled residue C46 [9]. Cyclic-AMP-dependent protein kinase phosphorylated residues C87 and ClOO [8], while C100 was also a target for the calmodulin-dependent multiprotein kinase [3, 41. The in vivo phosphorylation state of glycogen synthase is under hormonal control, increasing in response to adrenalin (decreasing activity) and decreasing in response to insulin (increasing activity) [l 11. In normally fed animals injected with the P-adrenergic antagonist propranolol, the alkali-labile phosphate bound covalently to glycogen synthase was found to be 2.9 mol/inol subunit and this value increased to 5.1 mol/ mol subunit in animals injected with adrenalin [12]. In animals deprived of food for ...