Three days after denervation, the expression of GLUT4 mRNA and protein decreases by approximately 50% in rat hindlimb muscles, while GLUT1 mRNA increases transiently by approximately 500%. Although postreceptor insulin resistance of glucose transport develops before GLUT4 declines, the latter likely contributes to the time-dependent increased severity of the resistance. To determine whether muscle inactivity contributes to changes in glucose transporter expression, one rat hindlimb was immobilized in a plaster cast for 3 days; the unencumbered hindlimb served as control. Muscle GLUT4 mRNA decreased by 32% (P < .02) and GLUT4 protein by 40% (P < .05) after 3 days' immobilization. There was no significant change in GLUT1 mRNA or skeletal muscle alpha-actin mRNA expression or in the total RNA concentration. The data suggest that electromyogenic and/or contractile activity regulates GLUT4 expression in skeletal muscle at a pretranslational step.