The metabolism of [1,2–¹³C₂] acetate via the tricarboxylic acid (TCA) cycle leads to the formation of a number of key mass isotopomers of glutamate. The distribution of these isotopomers which is a function of pyruvate carboxylase, pyruvate dehydrogenase and pyruvate recycling was used to determine the relative anaplerotic flux and glutamine oxidation of astrocytes in culture under different substrate conditions. Combinatory analysis of mass isotopomers formed from the condensation of labeled oxaloacetate with labeled acetyl-CoA was used to determine precursor enrichment and fractional glutamine synthesis. When glucose or glutamine was supplied in the medium, the effective anaplerotic flux (Y') was about 1.5 times that of the TCA cycle flux. Under substrate-limiting conditions, Y' and glutamine synthesis was significantly reduced. A unique feature of the use of [1, 2–¹³C₂] acetate in this study is the formation of singly labeled isotopomer of glutamine in the C4 or C5 position when glutamine is irreversibly loss in net oxidation. We observed very little [4-¹³C] or [5-¹³C] glutamine either because of the lack of pyruvate recycling or the lack of pyruvate dehydrogenase activity. The lack of ^l3C recycling to the C4 and C5 position of glutamine suggests that less than 10% of the glutamine is oxidized in astrocytes for energy production. Therefore, glutamine is not a major energy substrate for astrocytes in culture.