The ability of cultured astrocytes to metabolize [U-13C]glutamate in the absence of glucose was investigated by utilizing 13C nuclear magnetic resonance spectroscopy to identify 13C-labeled metabolites. Control cultures (3 mM glucose), hypoglycemic cultures (glucose-deprived), severe hypoglycemic cultures (glucose-deprived, 0.5 mM iodoacetate as an inhibitor of glycolysis), hypoglycemic/hypoxic cultures, and cultures deprived of all additional substrates were incubated for 2 hr in medium containing 0.5 mM glutamate (50% [U-13C]glutamate). Glucose deprivation alone had little effect on removal of glutamate from the culture medium, but the presence of iodoacetate or incubating cultures in a low-oxygen atmosphere decreased glutamate clearance. Only the withdrawal of all substrates other than glutamate decreased glutamine synthesis. Metabolism of glutamate through the tricarboxylic acid (TCA) cycle was evident by the appearance of [1,2,3-13C]glutamate and [U-13C]aspartate in cell extracts and [U-13C]lactate in cell media. Lactate derived from TCA cycle intermediates was significantly reduced after glucose deprivation and even more so after severe hypoglycemia. Release of glutamate from astrocytes was observed under all incubation conditions. [U-13C]Aspartate was not detected in control media but was released from glucose-deprived cells when oxygen was available. Increased release was observed in the presence of iodoacetate. After withdrawal of all substrates other than glutamate, [U-13C]aspartate was the only metabolite observed intracellularly, whereas aspartate, glutamine, and 5-oxoproline were detected in the incubation medium. The present results indicate that glutamate-to-aspartate conversion is preferentially utilized by astrocytes when oxygen is available but glycolysis is impaired.