It is not known whether the insulin-induced changes in the skeletal muscle glucose transport system occur under physiological circumstances. To clarify whether, by which mechanisms, and for how long skeletal muscle glucose transport activity is increased after an oral glucose load (OGL), we prepared plasma membrane (PM) and microsomal membrane (MsM) vesicles from hindlimb muscles of Sprague-Dawley rats either in the fasting state or 30, 60, 90, or 120 min after an OGL (2 g/kg body wt). In both PM and MsM, we measured the total number of glucose transporters (Ro), GLUT4, and GLUT1. In the PM, we also determined glucose influx (Vmax) and carrier turnover number (TN), an index of average transporter intrinsic activity, (TN = Vmax/Ro). The Vmax significantly increased after OGL, was maximal at 30 min, and returned to baseline at 90 min. The Ro and GLUT4 in the PM also increased significantly, with the maximum level reached at 60 min. The TN was increased only at 30 min. The changes in Ro and GLUT4 in the MsM were opposite to those in the PM, consistent with translocation of GLUT4 from an intracellular pool to the PM. In conclusion, an OGL induces an increase in the skeletal muscle glucose transport activity. This increase is associated with the translocation of GLUT4 from the MsM to the PM and a more transient increase in the average transporter TN. Our results show that transporter translocation and activation occur under physiological circumstances.