We have previously reported that autocrine secretion of insulin-like growth factor-II (IGF-II) plays a critical role in stimulating spontaneous myogenic differentiation in vitro. Myogenesis and IGF-II gene expression are both negatively controlled by high serum growth medium, and it is likely that serum inhibits terminal differentiation at least in part by blocking autocrine secretion of IGF-II. To investigate this possibility, we assessed the effects of various serum fractions and growth factors on endogenous IGF-II gene expression in rat L6A1 myoblasts. Unexpectedly, we found that IGF-I, IGF-II, and high concentrations of insulin were potent inhibitors of IGF-II gene expression. This is the first example we have seen in which IGFs regulate their own expression by a negative feedback mechanism. Feedback inhibition was not dependent on the stimulation of cell proliferation by IGFs, and differentiated L6A1 myotubes remained sensitive to this action of the IGFs. Results with IGF analogs suggested that the inhibition of IGF-II gene expression by IGFs was mediated by the type I IGF receptor and was strongly suppressed by L6A1-secreted IGF-binding proteins. Human primary myoblasts also exhibited feedback inhibition by the IGFs, whereas the rapidly fusing mouse Sol 8 cell line did not. We conclude that IGF-II gene expression in differentiating L6A1 myoblasts is regulated by a negative feedback mechanism (unusual for the IGFs) that acts primarily through the type I IGF receptor and appears to be inhibited by IGF-binding proteins secreted by L6A1 myoblasts in low serum differentiation medium.