The glycogen content of cultured chick embryo breast muscle cells changes during their development and can be reduced by starvation. It is demonstrated that the rate of glucose incorporation into glycogen and the degree of interconversion of glycogen synthase are controlled by the actual glycogen content. Stimulation of both corresponding activities by insulin is found in fused and in unfused cells. The insulin response depends on the extracellular calcium concentration and can be mimicked by the ionophore A 23187. These metabolic effects as well as calcium efflux data confirm the hypothesis that insulin acts on its enzyme target via increased cytoplasmic calcium concentration. Cytochalasin B is shown to inhibit the interconversion but does not interfere with the insulin-induced increase of the mitochondrial calcium pool or with the acceleration of the calcium efflux out of 45C-preloaded myotubes.