Glycogen synthase (GS) catalyses the regulatory step in glycogen synthesis by transferring glucose units from UDPGlucose to glycogen. This enzyme is regulated by multi-step reversible phosphorylation and allosteric modulation by the effector glucose-6-phosphate (G6P). Although many nucleotide sequences encoding GS have been isolated, only the rabbit and the yeast Gsy2 enzymes have their phosphorylation sites well characterized. Rabbit muscle enzyme has nine phosphorylation sites located both at the N- and C-terminus while the yeast enzyme has three C-terminus phosphorylation sites. Based on a sequence alignment of different GS enzymes the putative phosphorylation sites in GSN (706 amino acids) are located at C-terminus. Our main purpose is to identify the sites in GSN in order to evaluate the contribution of each site to the enzyme global regulation. Phosphorylation assays were performed by transferring γ-³²P-ATP to the protein using recombinant GSN, produced in E. coli fused to His tag, and crude cell extract of N. crassa strain RP-1 (gsn^rip) as source of protein kinases. Two GSN isoforms were used in the experiments: the entire protein (His-GSN) and a truncated form (His-GSN∆624) missed in the region containing the putative phosphorylation sites. Both GSN isoforms were phosphorylated in vitro indicating the existence of phosphorylation site(s) outside the C-terminus. As an attempt to identify the amino acids residues modified by phosphorylation, the isoforms were produced in the yeast Pichia pastoris, an eukaryotic organism, and thus able to perform posttranslational modification. Yeast strains producing the recombinant isoforms were isolated and cell extracts were fractionated in uni- and bidimensional SDS-PAGE gels. Regions containing the proteins were removed from the gels, trypsin digested and the phosphorylated peptides will be identified by mass spectrometry (Q-TOF).