The effect of thiopental on [U-(13)C]glutamate metabolism was studied in cerebellar astrocytes from mice using (13)C magnetic resonance spectroscopy. The cells were incubated with 0.5 mM [U-(13)C]glutamate for 2 h in the presence of 1 mM thiopental and 1 mM thiopental plus 0.2 mM gammaamino butyric acid (GABA). Labeled glutamate, glutamine, aspartate and glutathione were observed in cell extracts, and glutamine, aspartate and lactate in the media. Not only uniformly labeled glutamate was present in the medium, but also glutamate derived from the tricarboxylic acid (TCA) cycle. In the presence of thiopental, the amount of unlabeled glucose and [U-(13)C]glutamate removed from the medium was unchanged. This is in contrast to previous results obtained in cortical astrocytes, showing cellular heterogeneity. The concentrations of [1,2,3-(13)C]glutamate and [U-(13)C]glutamine were increased in the cell extracts, but unchanged in the medium, indicating an increased synthesis and an unchanged exchange or release. It should be noted that [U-(13)C]lactate is formed from [U-(13)C]glutamate via the TCA cycle and is released to the medium. In the presence of thiopental less [U-(13)C]lactate was observed in the medium. GABA had no influence on the effects of thiopental on cerebellar astrocytes.