After reaching confluence, mononucleated L6 myoblasts fuse into multinucleated contracting myotubes. This process is accompanied by the synthesis of characteristic skeletal muscle proteins, such as myosin heavy chain and the MM isoenzyme of creatine kinase. We have studied the development of insulin receptors and insulin responsiveness during differentiation in the L6 cells. Insulin was bound to high affinity receptors in both myoblasts and differentiated myotubes. The binding showed characteristics typical for insulin binding in other cell types, including high affinity, appropriate specificity, an upwardly concave Scatchard plot, and down-regulation. In the logarithmic growth phase, the myoblasts exhibited a low level of insulin binding, but on initiation of cell fusion, the resulting myotubes progressively developed a 2-fold increase in specific [125I]iodoinsulin binding as a result of a 2-fold increase in receptor number. The increase in insulin binding was an early differentiation event, preceding the accumulation of creatine kinase by 24 h. The development of insulin binding during differentiation correlated closely with an increased ability of the hormone to stimulate maximal 2-deoxy-D-glucose and alpha-aminoisobutyric acid uptake at physiological concentrations. The L6 cells are a useful model for studying the binding and effects of physiological insulin concentrations in skeletal muscle before and after differentiation.