Sleep is thought to be restorative in function, but what is restored during sleep is unclear. Here we tested the hypothesis that increased periods of wakefulness will result in decreased levels of glycogen, the principal energy store in brain, and with recovery sleep levels of glycogen will be replenished, thus representing a homeostatic component of sleep drive. Using a high-energy focused microwave irradiation method to kill animals and thereby snap-inactivate glycogen-producing and -metabolizing enzymes, we determined, with accuracy and precision, levels of brain glycogen and showed these levels to decrease significantly by ∼40% in brains of rats deprived of sleep for 12 or 24 hr. Recovery sleep of 15 hr duration after 12 hr of sleep deprivation reversed the decreases in glycogen. Using a novel histochemical method to stain brain glycogen, we found glycogen to be concentrated in white matter; this finding was confirmed biochemically in white matter dissected from rats killed with microwave irradiation. Levels of glycogen, as determined histochemically, were significantly decreased in gray and white matter with sleep deprivation, and these decreases were reversed with recovery sleep. The observed decreases in levels of brain glycogen may be a consequence of increased wakefulness and/or a component integral to the homeostatic drive to sleep.