The existence of metabolic compartmentation is generally accepted for the brain because of the specific differences in glial and neuronal metabolism. Extracellularly supplied acetate is believed to serve as a substrate only for glia cells, but not for neurons. To further test this hypothesis, primary rat brain cultures of neurons, astrocytes, and cocultures of both cell types were used to investigate the metabolic fate of [2-13C]acetate by means of nuclear magnetic resonance (NMR) spectroscopy. Acetate was metabolized by both cell types, neurons and astrocytes in culture. While glutamine was the main product of glial metabolism, labelled glutamate and aspartate were detected in neuron cultures. Astrocytes and neurons in cocultures showed a very different picture, which is similar to results obtained from brain slices, or in vivo studies. The analysis of the isotopomer pattern of glutamate and glutamine confirm an active glutamate-glutamine-cycle between neurons and astrocytes in cocultures. Furthermore, the analysis of unlabelled metabolites revealed a metabolic coupling of hypotaurine and taurine metabolism in astrocytes and neurons, which provides both cell types with one of the most important organic osmolytes.