Neurons in the ventromedial hypothalamus mediate some counterregulatory responses to hypoglycemia and 2-deoxyglucose, but the mechanisms that mediate these responses to glucose are unclear. In the present study, ventromedial hypothalamus neurons were identified on the basis of their inhibition by the transition from 5 to 20 mmol/l glucose. Tolbutamide, which activates glucose-stimulated neurons, failed to inhibit or activate glucose-inhibited neurons. Inhibitors of glucose transport and glycolysis, in particular by the glucokinase inhibitor glucosamine, blocked the effect of glucose on glucose-inhibited neurons. Furthermore, the glucose-inhibited neurons were activated by 2-deoxyglucose, which also activates counterregulatory responses. Conversely, glucose-inhibited neurons were inhibited by glycolytic metabolites, including lactate, but not by pyruvate. These data indicate that hypoglycemia induces electrical activity in glucose-inhibited neurons by attenuating glycolysis in those neurons. Thus, counterregulatory failure could be due to relatively enhanced glycolysis in glucose-stimulated neurons during hypoglycemia and attenuation of glycolysis in glucose-inhibited neurons might reverse counterregulatory failure.