To clarify the role of the ventromedial hypothalamus (VMH)-sympathetic nervous system in insulin-independent glucose uptake in peripheral tissues, tissue glucose uptake was assessed in vivo by the 2-[3H]deoxyglucose method during electrical stimulation of the VMH in anesthetized rats. VMH stimulation significantly increased the rate constant of glucose uptake in brown adipose tissue (BAT), heart and skeletal muscles, but not in white adipose tissue and brain. The effect of VMH stimulation on glucose uptake in BAT was abolished by local sympathetic denervation, indicating that the increase in glucose uptake is mediated by the sympathetic nerves. Electrical stimulation of the lateral hypothalamus, on the other hand, had no appreciable effects on 2-[3H]deoxyglucose uptake in any tissues. Changes in glucose transporters after VMH stimulation were also examined by the [3H]cytochalasin B binding method using sarcolemmal membranes isolated from heart muscle. Scatchard analysis of cytochalasin B binding indicated that VMH stimulation did not alter both the number and affinity (dissociation constant) of glucose transporters in the heart sarcolemmal membranes, whereas insulin administration increased the number of transporters in the membranes. These results suggest that the mechanism by which VMH stimulation increases glucose uptake in muscle is different from that of insulin.