AbstractCerebral glycogen is principally localized in astrocytes rather than in neurons. Glycogen metabolism has been implicated in higher brain functions, including learning and memory, yet the distribution patterns of glycogen in different types of astrocytes have not been fully described. Here, we applied a method based on the incorporation of 2-NBDG, a d-glucose fluorescent derivative that can trace glycogen, to investigate glycogen’s distribution in the brain. We identified two types of astrocytes, namely, 2-NBDG^I (glycogen-deficient) and 2-NBDG^II (glycogen-rich) cells. Whole-cell patch-clamp and fluorescence-activated cell sorting (FACS) were used to separate 2-NBDG^II astrocytes from 2-NBDG^I astrocytes. The expression levels of glycogen metabolic enzymes were analyzed in 2-NBDG^I and 2-NBDG^II astrocytes. We found unique glycogen metabolic patterns between 2-NBDG^I and 2-NBDG^II astrocytes. We also observed that 2-NBDG^II astrocytes were mainly identified as fibrous astrocytes but not protoplasmic astrocytes. Our data reveal cell type-dependent glycogen distribution and metabolism patterns, suggesting diverse functions of these different astrocytes.