Glial metabolism and their metabolic trafficking with neurons are essential parts of neuronal function, as they modulate, by this means, neuronal activity. Ex vivo and in vitro13C-NMR spectroscopy have been used to monitor neural cellular and tissue metabolism. Special emphasis has been given to the metabolic specialization of astrocytes and its enzymatic regulation. For this purpose primary cell cultures are useful tools to study neuronal–glial metabolic relationships as the extracellular fluid can be investigated and manipulated by various stimuli. In astrocytes, glucose is utilized predominantly anaerobically. Glycolysis is interrelated to the astrocytic TCA cycle via bi-directional signals and metabolic exchange processes between astrocytes and neurons. Besides glucose oxidation, neuronally released glutamate is metabolized through the glial TCA cycle. The flexibility of glutamate metabolism, depending on ammonia and energy homeostasis, and the discovered pyruvate recycling pathway in astrocytes, modulates the glutamine–glutamate cycle. 13C-NMR studies have extended the concept of the ‘non-stoichiometric’ glutamate–glutamine cycle between neurons and astrocytes. An alanine–lactate shuttle between neurons and astrocytes contributes to nitrogen transfer from neurons to astrocytes, recycles energy substrates for neurons, and in return promotes intercellular glutamine–glutamate cycling. The conversion of alanine to lactate in astrocytes is regulated by intracytosolic pyruvate compartmentation. In essence, the metabolic flexibility and compartmentalized enzymatic specialization of astrocytes buffers the brain tissue against metabolic impairments and excitotoxicity in response to extracellular stimuli, some of them being released by neurons. These in vitro studies using 13C-NMR spectroscopy provide important knowledge regarding physiological and pathophysiological regulation of neural metabolism to improve our understanding of general brain function. Copyright © 2003 John Wiley & Sons, Ltd.