The metabolism of [U-13C]aspartate was studied in cultured cortical astrocytes and cerebellar granule neurons in the presence of glucose and during inhibition of glycolysis. Redissolved, lyophilized cell extracts and incubation media were analyzed by 13C nuclear magnetic resonance spectroscopy for the determination of metabolites labeled from aspartate. Uniformly labeled lactate was prominent in control media of astrocytes and cerebellar granule neurons. In both cell types, aspartate entered the tricarboxylic acid (TCA) cycle, as shown by labeling patterns in glutamate and, in astrocytes, in glutamine. From the complex labeling patterns in aspartate in astrocytic perchloric acid extracts it was clear that acetylcoenzyme A (acetyl-CoA) derived from aspartate via oxaloacetate and pyruvate could enter the TCA cycle. Such "recycling," however, could not be detected in cerebellar granule neurons. Inhibition of glycolysis reduced aspartate uptake and metabolism in both cell types. Most notably, lactate derived from aspartate showed a large reduction, and in astrocytes, incorporation of labeled acetyl-CoA into the TCA cycle was significantly reduced. Thus, astrocytes and cerebellar granule neurons differ in their handling of aspartate. Furthermore, inhibition of glycolysis clearly affected aspartate metabolism by such cells.