The glutamate-glutamine cycle is thought to be of paramount importance in the mature brain; however, its significance is likely to vary with regional differences in distance between astrocyte and synapse. The present study is aimed at evaluating the role of this cycle in cultures of cerebellar neurons, mainly consisting of glutamatergic granule cells. Cells were incubated in medium containing [U-13C]glutamate or [U-13C]glutamine in the presence and absence of unlabeled glutamine and glutamate, respectively. Cell extracts and media were analyzed using high-performance liquid chromatography (HPLC) and gas chromatography combined with mass spectrometry (GC/MS). Both [U-13C]glutamate and [U-13C]glutamine were shown to be excellent precursors for synthesis of neuroactive amino acids and tricarboxylic acid (TCA) cycle intermediates. Labeling from [U-13C]glutamate was higher than that from [U-13C]glutamine in all metabolites measured. The presence of [U-13C]glutamate plus unlabeled glutamine in the experimental medium led to labeling very similar to that from [U-13C]glutamate alone. However, incubation in medium containing [U-13C]glutamine in the presence of unlabeled glutamate almost abolished labeling of metabolites. Thus, it could be shown that glutamate is the preferred substrate for intermediary metabolism in cerebellar neurons. Label distribution indicating TCA cycle activity showed more prominent cycling from [U-13C]glutamine than from [U-13C]glutamate. Labeling of succinate was lower than that of the other TCA cycle intermediates, indicating an active role of the gamma-amino butyric acid shunt in these cultures. It can be concluded that the cerebellar neurons rely more on reuptake of glutamate than supply of glutamine from astrocytes for glutamate homeostasis.