Sulfonylureas potentiate the insulin-induced stimulation of glucose transport in adipocytes. In order to elucidate the molecular mechanism of this potentiation, effects of a long-term (48-h) incubation with glyburide (2 micrograms/ml) on the relative sizes of the plasma membrane, the microsome, and the total membrane pools of the glucose-sensitive cytochalasin B binding were studied for basal and insulin-stimulated adipocytes. The drug treatment potentiated an insulin-induced stimulation of 3-O-methyl-D-glucose flux by 31 to 45%, with little effect on the basal flux. The same drug treatment increased the plasma membrane pool size of the glucose-sensitive cytochalasin B binding in the insulin-stimulated adipocytes with a concomitant decrease in the microsomal pool size. This effect was minimal, if any, in basal adipocytes. The drug treatment did not affect the total glucose-sensitive, cytochalasin B binding capacity of adipocyte membranes. These results indicate that the drug treatment increases the insulin-induced recruitment of the glucose carrier from the microsome to the plasma membrane by 27-31%. It is concluded that potentiation of the insulin-induced stimulation of the hexose transport by sulfonylureas is mainly due to a potentiation of the insulin-induced recruitment of the glucose carrier.