The insulin-like growth factors (IGFs) have dramatic and complex effects on the growth of many tissues and have been implicated in both the proliferation and differentiation of skeletal muscle cells. A detailed analysis of gene expression was performed in L6E9 myoblast cultures treated with IGF-I to dissect the early events leading to the stimulation of myogenic differentiation by this growth factor. A time course of transcript accumulation in confluent L6E9 myoblasts treated with defined media containing IGF-I revealed an initial transient decrease in myogenic factors, accompanied by an increase in cell cycle markers and cell proliferation. This pattern was reversed at later time points, when the subsequent activation of myogenic factors resulted in a net increase in structural gene expression and larger myotubes. The data presented here support the hypothesis that IGF-I activates proliferation first, and subsequently stimulates events leading to the expression of muscle-specific genes in myogenic cell cultures.