Skeletal muscle is the major site of insulin-stimulated glucose uptake, with the majority of glucose that enters muscle fibres being stored as glycogen. Insulin promotes dephosphorylation and activation of glycogen synthase (GS) by inactivating GSK3. Insulin also promotes glucose uptake and glucose-6-phosphate (G6P) production, which allosterically activates GS. However, the relative contribution of these two regulatory mechanisms in vivo is not well understood. We have recently shown that a knock-in mice carrying a mutated form of GSK3 that cannot be inactivated by insulin have normal rate of glycogen synthesis and glycogen content in skeletal muscle, suggesting that allosteric activation by G6P could be the major mechanism by which muscle GS is activated in vivo (1, 2). Using a mammalian expression system, we carried out a mutagenesis analysis using point mutants of arginine residues situated in the putative G6P binding region of GS. We identified one point mutation that completely abrogates G6P-mediated activation of GS without altering GS expression levels or insulin-mediated activation through dephosphorylation. To address the physiological role of the allosteric regulation of GS in the control of glycogen synthesis in vivo, a knock-in mouse carrying this mutation has been generated. The GS knock-in mice are viable and present a normal growth. They express the enzyme at a similar level than their wild type counterparts in different muscle types, in liver and heart. Their muscle GS is completely resistant to G6P-mediated activation and insulin administration results in the same level of PKB and GSK3 phosphorylation and GS desphosphoylation as the wild type mice. We are carrying out the full characterisation of these mice and providing direct genetic evidence of the major role that G6P allosteric activation plays in the maintenance of physiological glycogen levels in skeletal muscle.