This study was designed to examine the effect of insulin stimulation on glucose transport in rat skeletal muscle. Sarcolemmal vesicles (SL) were isolated from the gastrocnemius-plantaris and quadriceps muscles from insulin-stimulated and control groups. The insulin-stimulated group received an intravenous insulin injection (1 U/kg) 10 min before isolation. The early time course of specific D-glucose transport was linear through 2 s. Michaelis-Menten kinetics at 1.5 s indicated that the Vmax for glucose transport was increased after insulin stimulation compared with controls (4,424 +/- 668 vs. 1,366 +/- 124 pmol.mg protein -1.s-1), whereas the Km remained unchanged (19.4 +/- 0.6 vs. 21.6 +/- 3.1 mM). Scatchard plots for the D-glucose-inhibitable class of cytochalasin B binding sites indicated that insulin stimulation increased the number of binding sites in the SL vesicles (9.3 +/- 0.6 vs. 5.5 +/- 0.3 pmol/mg protein) without altering the Kd (48 +/- 3 vs. 46 +/- 3 nM). That the increase in Vmax was greater than the increase in cytochalasin B binding sites indicates that insulin stimulation caused an increase in the turnover rate of existing transport molecules as well as an increase in the total number of SL glucose transport molecules.