The mechanism by which the fructose analogue 2,5-anhydro-D-mannitol (2,5-AM) elicits feeding behavior was investigated by studying its metabolism and biochemical effects in liver. Thin-layer chromatography of liver extracts from rats given 2,5-AM containing 14C-labeled 2,5-AM showed that the analogue is phosphorylated in vivo with a time course that parallels the eating response. In vivo 31P nuclear magnetic resonance spectroscopy of rat liver during intravenous infusion of 2,5-AM and high-resolution nuclear magnetic resonance analyses of liver extracts showed that 2,5-AM is rapidly phosphorylated in liver, trapping hepatic phosphate and decreasing ATP, inorganic phosphate, and phosphate diesters. These changes occurred in a time frame in which the feeding response is elicited in conscious animals given the same dose of 2,5-AM by the same route. During an interval in which 2,5-AM increased eating, it also increased urinary uric acid excretion, implicating enhanced adenosine degradation in the reduction in hepatic ATP. These results provide the first direct evidence that changes in a high-energy phosphate-carrying compound in liver may provide a signal to initiate eating behavior.