The primary goal of this study was to establish whether inhibition of GABA synthesis was sufficient to induce network hyperexcitability in a rat hippocampal slice model comparable to that seen with GABA receptor blockade. We used field and intracellular recordings from the CA1 region of rat hippocampal slices to determine the physiological effects of blocking GABA synthesis with the convulsant, 3-mercaptoproprionic acid (MPA). We measured the rate of synthesis of GABA and glutamate in slices using 2-13C-glucose as a label source and liquid chromatography-tandem mass spectrometry. There was little effect of 3.5mM MPA on evoked events under control recording conditions. Tissue excitability was enhanced following a series of stimulus trains; this effect was enhanced when GABA transport was blocked. Evoked inhibitory potentials (IPSPs) failed following repetitive stimulation and MPA. Spontaneous epileptiform activity was seen reliably with elevated extracellular potassium (5mM). GABA synthesis decreased by 49% with MPA alone and 45% with the combination of MPA and excess potassium; GABA content was not substantially altered. Our data indicate: (1) GABAergic inhibition cannot be significantly compromised by MPA without network activation; (2) GABAergic synaptic inhibition is mediated by newly synthesized GABA; (3) there is a depletable pool of GABA that can sustain GABAergic inhibition when synthesis is impaired under basal, but not activated conditions; (4) overt hyperexcitability is only seen when newly synthesized GABA levels are low.