To study the mechanism of acceleration of glucose transport in skeletal muscle after stimulation with insulin and contractions, we isolated a subcellular vesicular membrane fraction, highly enriched in the plasma membrane enzyme K(+)-stimulated p-nitrophenylphosphatase and also enriched in some intracellular membranes. Protein recovery, morphology, lipid content, marker enzyme activities, total intravesicular volume, Western blot quantitation of GLUT-1, and glucose-inhibitable cytochalasin B binding were identical in membrane fractions from control, insulin-stimulated, contraction-stimulated, and insulin- and contraction-stimulated muscle. Time course of D-[3H]glucose entry in membrane vesicles at equilibrium exchange conditions showed that initial rate of transport at 30 mM of glucose was increased 19-fold and that equilibrium distribution space was increased 4-fold in vesicles from maximum stimulated muscle. The effects of insulin and contractions on initial rate of transport as well as on equilibrium distribution space were additive, and stimulation increased the substrate saturability of glucose transport. Furthermore, cytochalasin B binding to membranes prepared by using less centrifugation time than usual showed that, after stimulation with insulin and contractions, at least 35% of the total number of glucose transporters were redistributed from one kind of vesicles to a more slowly sedimenting kind of vesicles, probably reflecting translocation within the membrane preparation from intracellular vesicles to the plasma membrane upon stimulation. In the present membrane preparation the effects of insulin and/or contractions on glucose transport resemble those seen in intact muscle, and the effects are thus not dependent on cellular integrity.(ABSTRACT TRUNCATED AT 250 WORDS)