1. The influence of muscle glycogen content on glycogen synthase (GS) localization and GS activity was investigated in skeletal muscle from male Wistar rats. 2. Two groups of rats were obtained, preconditioned with a combination of exercise and diet to obtain either high (HG) or low (LG) muscle glycogen content. The cellular distribution of GS was studied using subcellular fractionation and confocal microscopy of immunostained single muscle fibres. Stimulation of GS activity in HG and LG muscle was obtained with insulin or contractions in the perfused rat hindlimb model. 3. We demonstrate that GS translocates from a glycogen-enriched membrane fraction to a cytoskeleton fraction when glycogen levels are decreased. Confocal microscopy supports the biochemical observations that the subcellular localization of GS is influenced by muscle glycogen content. GS was not found in the nucleus. 4. Investigation of the effect of glycogen content on GS activity in basal and insulin- and contraction-stimulated muscle shows that glycogen has a strong inhibitory effect on GS activity. Our data demonstrate that glycogen is a more potent regulator of glycogen synthase activity than insulin. Furthermore we show that the contraction-induced increase in GS activity is merely a result of a decrease in muscle glycogen content. 5. In conclusion, the present study shows that GS localization is influenced by muscle glycogen content and that not only basal but also insulin- and contraction-stimulated GS activity is strongly regulated by glycogen content in skeletal muscle.