Skeletal muscle responds to exercise by increasing the rate of glucose uptake. Recent studies have indicated that these changes occur via mechanisms modulating the number of transporters in the plasma membrane and/or transporter intrinsic activity. In the present study, a protocol was developed for measuring the initial rate of glucose uptake by rat hindlimb skeletal muscle plasma membrane vesicles. Membranes were isolated from sedentary (control) and acutely exercised rats, and the initial rates of D- and L-glucose influx were assayed under equilibrium exchange conditions to obtain the kinetic constants for carrier-mediated transport. These values were compared with the values for transporter number measured by cytochalasin B binding, and the carrier turnover numbers were calculated. The maximum velocity (Vmax) for carrier-mediated glucose influx was increased 3.7-fold by exercise, from 3.5 nmol.mg protein-1.s-1 for the membranes from control rats to 13 nmol.mg protein-1.s-1 for the membranes from exercised animals. The mean affinity constant (K0.5; approximately 20 mM) was not different between the two groups. The number of transporters in the plasma membrane was increased to a lesser degree, 5.4 to 9.4 pmol/mg protein. As a result, the average carrier turnover number was increased almost twofold by exercise, 719 s-1 in the controls vs. 1,380 s-1 in the exercised rats. These data indicate that the response of glucose transport to exercise involves an increase in the average carrier intrinsic activity as well as a recruitment of transporters to the plasma membrane. Whether the increase in carrier turnover number is due to activation of the transporters or recruitment of a more "active" form of the carrier is unknown.