The activation of muscle-specific myosin synthesis and its relationship to withdrawal from the cell cycle have been examined in cycle-synchronized myoblasts under growth-restrictive, fusion-impermissive (low Ca2+) culture conditions. Under these conditions, embryonic quail skeletal myoblasts, collected in mitosis by mechanical shake-off, complete one normal cycle and arrest in G1. The presence of skeletal muscle myosin is first detected, by indirect immunofluorescence, 8 hr into this protracted G1. Within the next 10-11 hr the percentage myosin positive (Myo+) cells increases with good synchrony, reaching approximately 95%. Refeeding with a proliferation--stimulating, low Ca2+ medium when approximately 50% of the cells are Myo+ induces reentry into S. Applying a 15-min pulse with [3H]TdR immediately preceding fixation at regular intervals following refeeding, cells can be detected which are Myo+ and whose nuclei have incorporated [3H]TdR. The numbers of such doubly labeled cells are small but consistent with the fraction of cells in S (by time-lapse analysis) at the postfeeding times sampled. These cinematographic studies also indicate that progression to mitosis following stimulation occurs slowly and asynchronously. The kinetics of progression of the stimulated cells suggest that they reenter S from a different compartment in G1 than do log-phase myoblasts. We conclude that in fusion-blocked quail myocytes irreversible withdrawal from the cell cycle is neither an obligate precondition for, nor an immediate consequence of the activation of the muscle-specific contractile gene set.