The effect of aminooxyacetic acid (AOAA), NH4+, phenylsuccinate (Phs), ketone bodies (KB) and glutamine (Gln), that might interfere with the biosynthesis of neurotransmitter glutamate on the K(+)-evoked Ca(2+)-dependent release of D-[3H]aspartate from rat cerebellar slices was studied. Therefore slices were preincubated in a Krebs-Ringer-bicarbonate-glucose (KR) buffer, loaded with D-[3H]aspartate and superfused in the presence of Ca2+ or when Ca2+ was replaced by Mg2+ or in some cases by EGTA. AOAA, NH4+ and Phs increase the K(+)-evoked Ca(2+)-dependent release of radioactivity by 30%, 68% and 188% compared to the control respectively indicating that these agents are inhibitors of the K(+)-evoked Ca(2+)-dependent release of glutamate. KB and Gln had no effect on the Ca(2+)-dependent release of radioactivity. AOAA, NH4+, Phs and KB but not Gln increase the total release of radioactivity by 43%, 69%, 139%, and 37% respectively. AOAA, NH4+ and KB but not Phs or Gln increase the Ca(2+)-independent release (Mg2+ replacing Ca2+) of radioactivity by 71%, 71% and 108% respectively. The present results indicate that in the cerebellum: 1) Neurotransmitter glutamate is mostly synthesized through the phosphate activated glutaminase (PAG) reaction 2) It is further supported that glutamate released in Ca(2+)-dependent manner before entering its pool in the cytosol has to move into the mitochondrial matrix.