Pyruvate carboxylase is the predominant anaplerotic enzyme in CNS tissues, and thus provides for net utilization of glucose to generate citric acid cycle intermediates such as alpha-ketoglutarate and malate for replenishment of the neurotransmitter pools of glutamate, GABA and aspartate. Studies reported in this paper involving immunocytochemical and biochemical techniques demonstrate: (1) the enzyme is localized in astrocytes as visualized by immunofluorescence in sections of cerebellum and (2) the enzyme activity in astrocyte-enriched populations is 3 X higher than in granule cell-enriched populations isolated from the cerebellum; similarly activity in different synaptosomal preparations parallels that for glutamine synthetase. We conclude from these results that the enzyme pyruvate carboxylase is an astrocyte-specific marker. This localization substantiates some recent hypotheses for astrocyte functions, including CO2 fixation in the CNS and the replenishment of citric acid cycle intermediates by astrocytes as precursors for amino acid neurotransmitter pools.