Hypothalamic malonyl-CoA has been shown to function in global energy homeostasis by modulating food intake and energy expenditure. Little is known, however, about the regulation of malonyl-CoA concentration in the central nervous system. To address this issue we investigated the response of putative intermediates in the malonyl-CoA pathway to metabolic and endocrine cues, notably those provoked by glucose and leptin. Hypothalamic malonyl-CoA rises in proportion to the carbohydrate content of the diet consumed after food deprivation. Malonyl-CoA concentration peaks 1 h after refeeding or after peripheral glucose administration. This response depends on the dose of glucose administered and is blocked by the i.c.v. administration of an inhibitor of glucose metabolism, 2-deoxyglucose (2-DG). The kinetics of change in hypothalamic malonyl-CoA after glucose administration is coincident with the suppression of phosphorylation of AMP kinase and acetyl-CoA carboxylase. Blockade of glucose utilization in the CNS by i.c.v. 2-DG prevented the effects of glucose on 5'AMP-activated protein kinase, malonyl-CoA, hypothalamic neuropeptide expression, and food intake. Finally, we showed that leptin can increase hypothalamic malonyl-CoA and that the increase is additive with glucose administration. Leptin-deficient ob/ob mice, however, showed no defect in the glucose- or refeeding-induced rise in hypothalamic malonyl-CoA after food deprivation, demonstrating that leptin was not required for this effect. These studies show that hypothalamic malonyl-CoA responds to the level of circulating glucose and leptin, both of which affect energy homeostasis.