Insulin causes a translocation of glucose transporters from intracellular microsomes to the plasma membrane in adipocytes. To determine whether insulin has a similar effect in rat hindlimb muscles, we used glucose-inhibitable cytochalasin B binding to estimate the number of glucose transporters in membrane fractions from insulinized and control muscles. Insulin treatment caused an approx. 2-fold increase in cytochalasin B-binding sites in a plasma membrane fraction and an approx. 70% decrease in cytochalasin B-binding sites in an intracellular membrane fraction. In order to detect this effect of insulin, it was necessary to develop a procedure for isolating a plasma membrane fraction and an intracellular membrane fraction that were not contaminated with sarcoplasmic reticulum. Our results show that, as in adipocytes, insulin stimulates translocation of glucose transporters from an intracellular membrane pool to the plasma membrane in hindlimb skeletal muscles.