Acute exercise, like insulin, increases D-glucose uptake into rat hind limb muscles. Here we examine the distribution of the muscle glucose transporters GLUT-4 and GLUT-1 in plasma membrane and intracellular membrane fractions of skeletal muscle prepared from control, exercised, and acutely insulin-treated rats. Immunoblotting with an anti-GLUT-4 polyclonal antibody showed that acute insulin treatment (by hind limb perfusion or in vivo injection) increased GLUT-4 transporters in a plasma membrane fraction and decreased them in an intracellular membrane fraction. Exercise also increased the GLUT-4 transporters in the plasma membrane, but in contrast to insulin, did not significantly decrease them in the intracellular fraction. Immunoblotting with anti-GLUT-1 antibody revealed that this transporter is largely localized in the plasma membrane. Neither insulin nor exercise significantly increased GLUT-1 transporters in the plasma membrane. The data show that GLUT-4 is an insulin-responsive glucose transporter in skeletal muscle and, furthermore, that GLUT-4 also responds to acute exercise. The results are consistent with recruitment of GLUT-4 glucose transporters to the plasma membrane from intracellular stores. Moreover, exercise-sensitive GLUT-4 transporters do not originate from the insulin-sensitive intracellular membrane fraction, suggesting the existence of distinct intracellular insulin- and exercise-recruitable GLUT-4 transporter pools.