Primary cultures of cerebral cortical astrocytes and neurons, as well as neurons growing on top of the astrocytes (sandwich co-cultures), were incubated with 1-[13C]glucose or 2-[13C]acetate and in the presence or absence of the glutamine synthetase inhibitor methionine sulfoximine. [13C]NMR spectroscopy at 125 MHz was performed on perchloric acid extracts of the cells or on media collected from the cultures. In addition, the [13C/12C] ratios of the amino acids glutamine, glutamate and 4-aminobutyrate (GABA) were determined by gas chromatography/mass spectroscopy, showing a larger degree of labeling in GABA than in glutamate and glutamine from glucose. Glutamine and glutamate were predominantly labeled from acetate. A picture of cellular metabolism mainly regarding the tricarboxylic acid cycle and glycolysis was obtained. Due to the fact that acetate is not metabolized by neurons to any significant extent, it could be shown that precursors from astrocytes are incorporated into the GABA pool of neurons grown in co-culture with astrocytes. Spectra of media removed from these cultures revealed that likely precursor candidates for GABA were glutamine and citrate. The importance of glutamine is further substantiated by the finding that inhibition of glutamine synthetase, an enzyme present in astrocytes only, significantly decreased the labeling of GABA in co-cultures incubated with 2-[13C]acetate.