Primary cultures of mouse cerebral cortical neurons (GABAergic) were incubated for 4 hours in media without glucose containing 1.0 mmol/L [U-13C]lactate in the absence or presence of 0.5 mmol/L glutamine. Redissolved, lyophilized cell extracts were analyzed by 13C nuclear magnetic resonance spectroscopy to investigate neuronal metabolism of lactate and by HPLC for determination of the total amounts of glutamate (Glu), gamma-aminobutyric acid (GABA), and aspartate (Asp). The 13C nuclear magnetic resonance spectra of cell extracts exhibited multiplets for Glu, GABA, and Asp, indicating pronounced recycling of labeled tricarboxylic acid cycle constituents. There was extensive incorporation of 13C label into amino acids in neurons incubated without glutamine, with the percent enrichments being approximately 60% for Glu and Asp, and 27% for GABA. When 0.5 mmol/L glutamine was added to the incubation medium, the enrichments for Asp, Glu, and GABA were 25%, 35%, and 25%, respectively. This strongly suggests that glutamine is readily converted to Glu and Asp but that conversion to GABA may be complex. The observation that enrichment in GABA was identical in the absence and presence of glutamine whereas cycling was decreased in the presence of glutamine indicates that only C-2 units derived from glutamine are used for GABA synthesis, that is, that metabolism through the tricarboxylic acid cycle is a prerequisite for GABA synthesis from glutamine. The current study gives further support to the hypothesis that cellular metabolism is compartmentalized and that lactate is an important fuel for neurons in terms of energy metabolism and extensively labels amino acids synthesized from tricarboxylic acid cycle intermediates (Asp and Glu) as well as the neurotransmitter in these neurons (GABA).