Mitogens are generally thought to inhibit myogenesis, and many cell biologists have found it hard to interpret observations that the insulin-like growth factors (IGFs) stimulate both proliferation and differentiation of muscle cells in culture. Our previous studies suggested that the Type I IGF receptor mediates these actions. However, IGF-II and insulin treatment caused myoblasts to differentiate much more extensively, suggesting that more complex mechanisms may be involved. Here we present evidence that the greater mitogenic activity of IGF-I (compared to IGF-II and insulin) delays L6A1 myoblast differentiation. Under conditions in which the mitogenic actions of IGF-I are suppressed, the stimulation of myogenesis by IGF-I approached that by IGF-II: (1) in L6A1 cultures plated at a higher cell density; (2) in L6A1 cultures in which cell proliferation was inhibited by cytosine arabinoside or aphidicolin; and (3) in cultures of primary human muscle cells, which exhibit a smaller mitogenic response to IGF-I. Further evidence that the Type I receptor plays a major role in relaying the signal for differentiation was obtained by using IGF-I and IGF-II analogs. Analogs which have reduced affinity for the Type I receptor showed a dramatic decrease in activity, while an analog with increased affinity for the Type II receptor was no more active than native IGF-I. Our results indicate that both mitogenic and myogenic actions of IGF-I are mediated by the Type I receptor. We conclude that IGF-I delays the onset of myogenesis as a result of its mitogenic actions, and only subsequently stimulates myogenesis. These observations reconcile the apparent conflict between our results with the IGFs and other investigators' reports of effects of other mitogens.