Slices of the molecular layer of the dentate gyrus of the hippocampal formation were incubated with either [14C]glucose, [14C]pyruvate or 14C glutamine and the efflux of endogenous and radioactive glutamate was monitored under various conditions. After prelabeling with either [14C]glutamine or [14C]glucose elevation of K+ concentration to 56 mM (Ca2+ free) increased efflux of endogenous and [14C]glutamate. Introduction of Ca2+ into the elevated K+ medium further increased the efflux of endogenous glutamate and radioactive glutamate derived from any of the precursors tested. In glutamine containing media, the increase in glutamate efflux as well as basal efflux was considerably higher than in the absence of glutamine and the specific activity of glutamate release was higher than that in tissue. Thus glutamine was superior to glucose or pyruvate as precursor and most specifically labeled the putative transmitter pool of glutamate. Similar experiments were carried out 4 and 14 days after a unilateral lesion in the entorhinal cortex which provides about 60% of the total synaptic input to the dentate granule cells. The Ca2+ dependent release of glutamate derived from either glucose or glutamine was markedly reduced on the operated side. This result suggests that the transmitter pool of glutamate is in perforant path terminals and can be synthesized from glucose or glutamine.