Skeletal muscle contractile activity results in increased rates of glucose transport that are associated with an increase in the number and activity of plasma membrane glucose transporters. In the current study it was determined whether exercise causes a translocation of glucose transporters from an intracellular pool to the plasma membrane and whether exercise and insulin stimulate the same glucose transporter protein. Plasma membrane glucose transporter number, measured by cytochalasin B binding, increased from 10.1 +/- 0.73 to 15.0 +/- 1.4 pmol/mg protein (P less than 0.01) in muscle of exercised rats, whereas microsomal membrane transporters decreased significantly from 6.0 +/- 0.7 to 4.2 +/- 0.4 pmol/mg protein (P less than 0.05). Western blot analysis using the monoclonal antibody mAb 1F8 (specific for GLUT-4) demonstrated a 45% increase in plasma membrane GLUT-4 from exercised skeletal muscle compared with controls, whereas microsomal membranes from the exercised muscle had a concomitant 25% decrease in GLUT-4 protein. These data suggest that exercise recruits transporters to the plasma membrane from an intracellular microsomal pool, similar to the translocation of transporters that occurs with insulin stimulation. Furthermore, both exercise and insulin stimulate the translocation of GLUT-4 in skeletal muscle, while GLUT-1 is not altered.