Nutrient-sensitive hypothalamic neurons regulate energy balance and glucose homeostasis, but the molecular mechanisms mediating hypothalamic responses to nutritional state remain incompletely characterized. To address these mechanisms, the present studies used quantitative PCR to characterize the expression of a panel of genes the hypothalamic expression by nutritional status of which had been suggested by DNA microarray studies. Although these genes regulate a variety of function, the most prominent set regulate intermediary metabolism, and the overall pattern clearly indicated that a 48-h fast produced a metabolic reprogramming away from glucose metabolism and toward the utilization of alternative fuels, particularly lipid metabolism. This general reprogramming of intermediary metabolism by fasting was observed both in cortex and hypothalamus but most prominently in hypothalamus. The effect of fasting on the expression of these genes may be mediated by reduction in plasma glucose or glucose metabolism, rather than leptin, because they were generally recapitulated by hypoglycemia even in the presence of elevated insulin and in vitro by low glucose but were not recapitulated in ob/ob mice. These studies suggest that fasting reduces glucose metabolism and thus minimizes the production of hypothalamic malonyl-coenzyme A. However, because the reprogramming of glucose metabolism by fasting was also observed in cortex, this apparent substrate competition may mediate more general responses to nutritional deprivation, including those responsible for the protective effects of dietary restriction. The present studies also provide a large panel of novel glucose-regulated genes that can be used as markers of glucose action to address mechanisms mediating hypothalamic responses to nutritional state.