The fiber type composition of a skeletal muscle is an important determinant of its ability to take up glucose. Although numerous factors may account for this phenomenon, we have hypothesized that fiber type differences in glucose transporter number, isoform content, and/or intrinsic activity play an important role. Skeletal muscle plasma membranes were prepared from red and white gastrocnemius muscle from male Sprague-Dawley rats that were either exercised on a treadmill (1 h, 20 m/min, 10% grade), injected with 20 U insulin, or remained sedentary. In sedentary rats, plasma membrane glucose transporter number (cytochalasin B binding) was 2.4-fold greater in red compared with white muscle. Exercise and insulin both increased glucose transporter number by 40% in red muscle and twofold in white muscle. Maximal velocity of glucose transport (Vmax) was 2-fold greater in red compared with white muscle, whereas exercise and insulin increased Vmax by 2.3-fold in red muscle and 3.6-fold in white muscle. Glucose transporter turnover number, a measure of the average intrinsic activity of transporters in the plasma membrane, was not different between red and white muscle and increased 80-90% with exercise and insulin in both red and white muscle. Both GLUT-1 and GLUT-4 isoform content were greater in red than white muscle. These results suggest that fiber type differences in rates of glucose uptake in resting, insulin-stimulated, and contraction-stimulated skeletal muscle may be due to differences in the number but not the intrinsic activity of glucose transporter proteins.