Spreading depression severely disrupts ion homeostasis, causes sensory neglect and motor impairment, and is associated with stroke and migraine. Glucose utilization (CMRglc) and lactate production rise during spreading depression, but the metabolic changes in different brain cell types are unknown. Uptake of 14C-labeled compounds known to be preferentially metabolized by the glial tricarboxylic acid cycle was, therefore, examined during unilateral KCl-induced spreading cortical depression in conscious, normoxic rats. [14C]Metabolites derived from [14C]butyrate in K+-treated tissue rose 21% compared to that of untreated contralateral control cortex, whereas incorporation of H14CO3 into metabolites in K+-treated tissue was reduced to 86% of control. Autoradiographic analysis showed that laminar labeling of cerebral cortex by both 14C-labeled acetate and butyrate was elevated heterogeneously throughout cortex by an average of 23%; the increase was greatest (∼40%) in tissue adjacent to the K+ application site. Local uptake of acetate, butyrate, and deoxyglucose showed similar patterns, and monocarboxylic acid uptake was highest in the structures in which apparent loss of labeled metabolites of [6-14C]glucose was greatest. Enhancement of net uptake of acetate and butyrate in cerebral cortex during spreading depression is tentatively ascribed to increased astrocyte metabolism. © 2001 Wiley-Liss, Inc.