Skeletal muscle differentiation is normally accompanied by the permanent withdrawal of myogenic nuclei from the proliferative cycle. However, embryonic Japanese quail (Coturnix coturnix japonica) myoblasts which have been prevented from fusing in vitro by the addition of EGTA to the culture medium retain the capacity to re-enter the cell cycle following accumulation of muscle-specific myosin. We have therefore investigated the roles of Ca2+ and fusion in the withdrawal of myogenic cells from the cell cycle. Using three defined media which differ in Ca2+ and in the ability to promote fusion, we examined the ability of differentiation-competent myoblasts to resume proliferation with increased time in G1. Under these conditions, there is a periodic variation in the ability of the myoblasts to respond to mitogenic stimulation, irrespective of the medium employed. These results indicate that loss of proliferative capacity during myogenesis is independent of Ca2+.