To study the effect of agents interfering with the biosynthesis and/or the K(+)-evoked Ca(2+)-dependent release of neurotransmitter glutamate, rat cerebral slices were preincubated with Krebs-Ringer-HEPES-glucose-glutamine buffer (KRH buffer), loaded with D-[3H]aspartate and superfused with the preincubation medium in the presence or in the absence of Ca2+. The difference in radioactivity release divided by the basal release per min under the two conditions represented the K(+)-evoked Ca(2+)-dependent release. The agents used were: 1) Aminooxyacetic acid (AOAA), the inhibitor of transaminases, 2) Leucine (Leu), the inhibitor of phosphate activated glutaminase (PAG), 3) NH4+, the inhibitor of PAG, 4) Phenylsuccinic acid (Phs), the inhibitor of the mitochondrial ketodicarboxylate carrier, 5) ketone bodies, the inhibitors of glycolysis, 6) the absence of glutamine, the substrate of PAG. The results show that Leu, NH4+, Phs and the absence of Gln significantly increase the K(+)-evoked Ca(2+)-dependent release of radioactivity by 64%, 200%, 95% and 147% respectively, indicating that these agents are inhibitors of the K(+)-evoked Ca(2+)-dependent release of glutamate. Ketone bodies and AOAA had no effect. These results indicate that the major if not the exclusive biosynthetic pathway of neurotransmitter glutamate in rat cerebral cortex is through the PAG reaction and support a model for the pathway followed by neurotransmitter glutamate i.e. glutamate formed outside the inner mitochondrial membrane has to enter the mitochondrial matrix or is formed within it from where it can be extruded to supply the transmitter pool in exchange of GABA.