γ-Aminobutyric acid (GABA) synthesis in the brain is mediated by two major isoforms of glutamic acid decarboxylase, GAD 65 and GAD 67 . The contribution of these isoforms to GABA synthesis flux (V GAD ) is not known quantitatively. In the present study we compared V GAD in cortex of control and vigabatrin-treated rats under α-chloralose/70% nitrous oxide anesthesia, with total GAD activity and GAD isoform composition (GAD 65 and GAD 67 ) measured by enzymatic assay and quantitative immunoblotting. V GAD was determined by re-analysis of 13 C NMR data obtained ex vivo and in vivo during infusions of [1- 13 C]glucose using an extension of a model of glutamate–glutamine cycling that included a discrete GABAergic neuronal compartment with relevant interconnecting fluxes. V GAD was significantly lower in vigabatrin-treated rats (0.030–0.05 μmol/min per g, P<0.003) compared to the non-treated control group (0.10–0.15 μmol/min per g). The 67–70% decrease in V GAD was associated with a 13% decrease in total GAD activity (P=0.01) and a selective 44±15% decrease in GAD 67 protein (from 0.63±0.10 to 0.35±0.08 μg protein/mg tissue, P<0.05); GAD 65 protein was unchanged. The reduction in GAD 67 protein could account for a maximum of ∼65% of the decrease in V GAD in vigabatrin-treated animals suggesting that inhibition of GAD 65 must have also occurred in these experiments, although product inhibition of GAD 67 by increased GABA could play a role. GAD 67 could account for 56–85% of cortical GABA synthesis flux under basal conditions and the entire flux after vigabatrin treatment.