The concentration of glycogen, the major brain energy reserve localized mainly in astrocytes, is generally reported as about 2 or 3 micromol/g, but sometimes as high as 3.9 to 8 micromol/g, in normal rat brain. The authors found high but very different glycogen levels in two recent studies in which glycogen was determined by the routine amyloglucosidase procedure in 0.03N HCl digests either of frozen powders (4.8 to 6 micromol/g) or of ethanol-insoluble fractions (8 to 12 micromol/g). To evaluate the basis for these discrepant results, glycogen was assayed in parallel extracts of the same samples. Glycogen levels in ethanol extracts were twice those in 0.03N HCl digests, suggesting incomplete enzyme inactivation even with very careful thawing. The very high glycogen levels were biologically active and responsive to physiologic and pharmacological challenge. Glycogen levels fell after brief sensory stimulation, and metabolic labeling indicated its turnover under resting conditions. About 95% of the glycogen was degraded under in vitro ischemic conditions, and its "carbon equivalents" recovered mainly as glc, glc-P, and lactate. Resting glycogen stores were reduced by about 50% by chronic inhibition of nitric oxide synthase. Because neurotransmitters are known to stimulate glycogenolysis, stress or sensory activation due to animal handling and tissue-sampling procedures may stimulate glycogenolysis during an experiment, and glycogen lability during tissue sampling and extraction can further reduce glycogen levels. The very high glycogen levels in normal rat brain suggest an unrecognized role for astrocytic energy metabolism during brain activation.