Adrenaline and insulin are the major hormones regulating glycogen metabolism in skeletal muscle. We have investigated the effects of these hormones on the rate-limiting enzymes of glycogen degradation and synthesis (phosphorylase and glycogen synthase respectively) in G_M^−/− mice homozygous for a null allele of the major skeletal muscle glycogen targeting subunit (G_M) of protein phosphatase 1 (PP1). Hyperphosphorylation of Ser14 in phosphorylase, and Ser7, Ser640 and Ser640/644 of GS, in the skeletal muscle of G_M^−/− mice compared with G_M^+/+ mice indicates that the PP1-G_M complex is the major phosphatase that dephosphorylates these sites in vivo. Adrenaline caused a 2.4-fold increase in the phosphorylase (−/+AMP) activity ratio in the skeletal muscle of control mice compared to a 1.4 fold increase in G_M^−/− mice. Adrenaline also elicited a 67% decrease in the GS (−/+G6P) activity ratio in control mice but only a small decrease in the skeletal muscle of G_M^−/− mice indicating that G_M is required for the full response of phosphorylase and GS to adrenaline. PP1-G_M activity and the amount of PP1 bound to GM decreased 40% and 45% respectively, in response to adrenaline in control mice. The data support a model in which adrenaline stimulates phosphorylation of phosphorylase Ser14 and GS Ser7 in G_M^+/+ mice by both kinase activation and PP1-G_M inhibition and the phosphorylation of GS Ser640 and Ser640/644 by PP1-G_M inhibition alone. Insulin decreased the phosphorylation of GS Ser640 and Ser640/644 and stimulated the GS (−/+G6P) activity ratio by ∼2-fold in the skeletal muscle of either G_M^−/− and or control mice, but the low basal and insulin stimulated GS activity ratios in G_M^−/− mice indicate that PP1-G_M is essential for maintaining normal basal and maximum insulin stimulated GS activity ratios in vivo.