Insulin and exercise cause the translocation of GLUT4 from an intracellular location to the plasma membrane in skeletal muscle. The purpose of this study was to determine if Rab4, a small GTP binding protein that has been implicated in the insulin-stimulated translocation of GLUT4 in adipose cells, is involved in the regulation of transporter translocation in skeletal muscle. Male rats were injected with insulin (20 U i.p.) or exercised on a treadmill(1 h, 20 m/min, 10% grade). Rats were killed 30 min after insulin injection or immediately after exercise, and the hind limb muscles dissected. Plasma membrane and intracellular microsomal membrane fractions were prepared, and the distribution of GLUT4 and Rab4 was determined by immunoblotting. Both insulin and exercise caused GLUT4 translocation as demonstrated by a decrease in microsomal membrane GLUT4 and an increase in plasma membrane GLUT4. In contrast, only insulin caused a decrease in Rab4 in the microsomal membrane. Rab4 was associated with GLUT4-containing vesicles isolated by immunoprecipitation. Rab4 was not detected in plasma membrane under any condition. These data demonstrate that insulin modulates the subcellular distribution of both GLUT4 and Rab4 in rats skeletal muscle, suggesting that Rab4 may play a role in the insulin-stimulated movement of GLUT4-containing vesicles. Although both insulin and exercise increase skeletal muscle glucose uptake by the translocation of GLUT4, the regulation of translocation may occur by different mechanisms.