The metabolism of glucose and lactate was investigated in cultured mouse cerebellar astrocytes, a culture preparation consisting of a homogeneous population of cells, by incubating the cells in a medium containing either [U-13C]glucose or [U-13C]lactate in combination with unlabeled lactate and glucose, respectively. After the incubation, cell extracts and media were analyzed by GC/MS (gas chromatography/mass spectrometry) for labeling patterns in aspartate, glutamate, and glutamine, as well as the tricarboxylic acid (TCA) cycle constituents citrate and fumarate. Triple labeling of extracellular citrate exceeded that of double labeling from [U-13C]glucose. This was not the case when lactate was the labeled precursor. These results indicate that pyruvate carboxylation in biosynthesis of releasable citrate was less prominent from lactate compared with glucose. As observed in the case of extracellular citrate, triple labeling of intracellular aspartate was higher than double labeling when [U-13C]glucose was the precursor, but not with [U-13C]lactate as precursor. The pattern of labeling in citrate was different intra- and extracellularly and the extent of labeling extracellularly was 10 times higher using [U-13C]glucose compared with [U-13C]lactate. However, the intracellular citrate labeling from [U-13C]glucose only exceeded that originating from labeled lactate by a factor of two. This is in contrast to the labeling pattern obtained for glutamine, since intracellularly this was equally prominent using [U-13C]glucose and [U-13C]lactate as substrates. Moreover, extracellularly the labeling was only slightly higher when using [U-13C]glucose compared with [U-13C]lactate. Intracellular glutamate and extracellular glutamine exhibited similar incorporation patterns with regard to double compared with triple labeling and the extent of incorporation of label from [U-13C]lactate compared with [U-13C]glucose. It should be noted that the main intracellular pools of citrate and glutamine were compartmentalized; i.e., releasable citrate and glutamine exhibited a labeling pattern distinctly different from that of their intracellular pools. Moreover, carboxylation of pyruvate using glucose as the precursor was more important for biosynthesis of releasable glutamine and citrate, compared with their intracellular pools. Based on the results a model of multiple compartments is suggested. The compartments differ with regard to utilization of lactate and glucose, synthetic pathways for TCA cycle intermediates and amino acids, particularly citrate and glutamine, as well as the contents of different metabolites. GLIA 35:246–252, 2001. © 2001 Wiley-Liss, Inc.