Skeletal myofibers differentiated from primary avian myoblasts in tissue culture can be maintained in positive nitrogen balance in a defined serum-free medium for at least 6-7 days when embedded in a three-dimensional collagen gel matrix. Incubation of established myofiber cultures for 3-7 days with insulin (1 microM) or insulin-like growth factor I (IGF-I, 32 nM) stimulates both cell hyperplasia and myofiber hypertrophy. Mean myofiber diameter increases 71-98%. Insulin-like growth factor II stimulates cell hyperplasia but not myofiber hypertrophy. Cell growth results from a 42-62% increase in total protein synthesis and a 28-38% decrease in protein degradation. Myosin heavy-chain content increases 183-258% because of a 55% stimulation of myosin synthesis and 33-61% inhibition of degradation. Associated with myofiber hypertrophy is a 87-148% increase in the number of myofiber nuclei per unit myofiber length. The results indicate that insulin and IGF-I, but not IGF-II, can induce rapid myofiber hypertrophy in vitro, most likely by stimulating myoblast proliferation and/or fusion to established myofibers.