Primary cultures of mouse cerebral cortical neurons were incubated for 3 h with 250 microM [U-13C]glutamate or 250 microM [U-13C]glutamine and 6 mM glucose. 13C NMR spectra of cell extracts exhibited distinct multiplets for glutamate, aspartate and GABA. Incorporation of label into aspartate can only occur through the tricarboxylic acid (TCA) cycle, but as demonstrated by formation of the 3,4-13C2-isotopomer of GABA and the 1,2,3-13C3-isotopomer of glutamate, these amino acids were also to some extent derived from this pathway. Formation of the 1,2-13C2-(1J1,2 = 50 Hz) and 3,4-13C2-isotopomer (1J3,4 = 50.9 Hz) in aspartate occurs exclusively when oxaloacetate (containing 12C) is derived from the second turn of the TCA cycle. From [U-13C]glutamine, the 12C containing isotopomers in aspartate accounted for 27% of the total label and from [U-13C]glutamate, it was less than 10%. When [U-13C]glutamine was the precursor, 36% of the labeled glutamate and 52% of the labeled GABA contained 12C incorporated during the first turn of the TCA cycle. These numbers decreased to 15 and 30%, respectively when [U-13C]glutamate was used. Since glutamine must be converted to glutamate before it can enter the TCA cycle as 2-oxoglutarate, appearance of 12C incorporation in aspartate should be identical when [U-13C]glutamate and [U-13C]glutamine was used as substrates. This was, however, not observed which may be indicative of (1) compartmentation of mitochondrial glutamate metabolism or (2) differences in the amount of intramitochondrial glutamate derived from external glutamate or glutamine.