The brain contains a small but significant amount of glycogen, which has long been considered to play an insignificant role in the brain. In this study, brain glycogen metabolism was measured using (13)C NMR spectroscopy at 9.4 T. Brain glycogen metabolism was modulated by hyperinsulinemia resulting in a net accumulation. The role of glycogen in maintaining brain function is unknown; one possibility is that it may serve as an endogenous glucose reservoir to protect the brain against severe hypoglycemia. To address this possibility, rats were subjected to insulin-induced moderate hypoglycemia and when the level of brain glucose approached zero, brain glycogen content began to decrease gradually, demonstrating utilization of this glucose reservoir. The brain glycogen signal never became undetectable, however, even during 2 hr of hypoglycemia. When plasma and brain glucose concentrations were restored, glycogen increased and the concentration exceeded the pre-hypoglycemic level by several-fold. The data suggest that brain glycogen can provide fuel for extended periods of time when glucose supply is inadequate. Furthermore, brain glycogen can rebound (super-compensate) after a single episode of hypoglycemia. We postulate that brain glycogen serves as an energy store during hypoglycemia and that it may participate in the creation of reduced physiological responses to hypoglycemia that are involved in a symptom often observed in patients with diabetes, hypoglycemia unawareness.