Muscle is a major tissue for insulin action. To study the effect of muscle differentiation on insulin receptors, we employed cultured mouse muscle BC3H-1 and C2 cells. In both cell lines differentiation from myoblasts to myocytes was associated with a 5-10-fold increase in specific 125I-insulin binding to intact cells. When 125I-insulin binding was carried out on solubilized myocytes and myoblasts, 125I-insulin binding to myoblasts was low. After differentiation the number of insulin receptors increased 5-10-fold. In contrast to insulin binding, insulin growth factor I receptor binding was elevated in myoblasts and was decreased by 50% in myocytes. Specific radioimmunoassay of the insulin receptor indicated that the increase in insulin binding to myocytes was due to an increase in insulin receptor content. Studies employing [35S]methionine indicated that this increase in insulin-binding sites reflected an increase in insulin receptor biosynthesis. To study insulin receptor gene expression, myoblast and myocyte mRNA was isolated and analyzed on Northern and slot blots. Differentiation from myoblasts to myocytes was accompanied by a 5-10-fold increase in insulin receptor mRNA. These studies demonstrate, therefore that differentiation in muscle cells is accompanied by increased insulin receptor biosynthesis and gene expression.