1. Cerebral-cortex slices prelabelled with γ-amino[1-14C]butyrate (GABA) were incubated in a glucose–saline medium. After the initial rapid uptake there was no appreciable re-entry of 14C into the GABA pool, either from the medium or from labelled metabolites formed in the tissue. The kinetic constants of GABA metabolism were determined by computer simulation of the experimental results by using mathematical procedures. The GABA flux was estimated to be 0.03μmol per min/g, or about 8% of the total flux through the tricarboxylic acid cycle. It was found that the assumption of compartmentation did not greatly affect the estimates of the GABA flux. 2. The time-course of incorporation of 14C into amino acids associated with the tricarboxylic acid cycle was followed with [1-14C]GABA and [U-14C]-glucose as labelled substrates. The results were consistent with the utilization of GABA via succinate. This was confirmed by determining the position of 14C in the carbon skeletons of aspartate and glutamate formed after the oxidation of [1-14C]GABA. These results also indicated that under the experimental conditions the reversal of reactions catalysed by α-oxoglutarate dehydrogenase and glutamate decarboxylase respectively was negligible. The conversion of [14C]GABA into γ-hydroxybutyrate was probably also of minor importance, but decarboxylation of oxaloacetate did occur at a relatively slow rate. 3. When [1-14C]GABA was the labelled substrate there was evidence of a metabolic compartmentation of glutamate since, even before the peak of the incorporation of 14C into glutamate had been reached, the glutamine/glutamate specific-radioactivity ratio was greater than unity. When [U-14C]glucose was oxidized this ratio was less than unity. The heterogeneity of the glutamate pool was indicated also by the relatively high specific radioactivity of GABA, which was comparable with that of aspartate during the whole incubation time (40min). The rates of equilibration of labelled amino acids between slice and medium gave evidence that the permeability properties of the glutamate compartments labelled as a result of oxidation of [1-14C]GABA were different from those labelled by the metabolism of [14C]glucose. The results showed therefore that in brain tissue incubated under the conditions used, the organization underlying metabolic compartmentation was preserved. The observed concentration ratios of amino acids between tissue and medium were also similar to those obtaining in vivo. These ratios decreased in the order: GABA>acidic acids>neutral amino acids>glutamine. 4. The approximate pool sizes of the amino acids in the different metabolic compartments were calculated. The glutamate content of the pool responsible for most of the labelling of glutamine during oxidation of [1-14C]GABA was estimated to be not more than 30% of the total tissue glutamate. The GABA content of the ‘transmitter pool’ was estimated to be 25–30% of the total GABA in the tissue. The structural correlates of metabolic compartmentation were considered.