Initial seizures observed in young rats during the 60 min after administration of pilocarpine (Pilo) were delayed and attenuated by pretreatment with a non-convulsive dose of methionine sulfoximine (MSO). We hypothesized that the effect of MSO results from a) glutamine synthetase block-mediated inhibition of conversion of Glu/Gln precursors to neurotransmitter Glu, and/or from b) altered synaptic Glu release. Pilo was administered 60 min prior to sacrifice, MSO at 75 mg/kg, i.p., 2.5 h earlier. [1,2-¹³C]acetate and [U-¹³C]glucose were i.p.-injected either together with Pilo (short period) or 15 min before sacrifice (long period). Their conversion to Glu and Gln in the hippocampus and entorhinal cortex was followed using [¹³C] gas chromatography-mass spectrometry. Release of in vitro loaded Glu surrogate, [³H]D-Asp from ex vivo brain slices was monitored in continuously collected superfusates. [³H]D-Asp uptake was tested in freshly isolated brain slices. At no time point nor brain region did MSO modify incorporation of [¹³C] to Glu or Gln in Pilotreated rats. MSO pretreatment decreased by ~37% high potassium-induced [³H]D-Asp release, but did not affect [³H]D-Asp uptake. The results indicate that MSO at a non-convulsive dose delays the initial Pilo-induced seizures by interfering with synaptic Glu-release but not with neurotransmitter Glu recycling.