Purified striatal synaptosomes were continuously superfused with L,3,5[3H]tyrosine in order to estimate the synthesis ([3H]water) and release of newly formed [3H]dopamine. In the presence of magnesium, L-glutamate, D,L-alpha-amino-3-hydroxy-5-methyl-4-isoxazole-4-propionate (AMPA) and kainate, but not N-methyl-D-aspartate (NMDA) and 1-aminocyclopentane-1S,3R-dicarboxylate (t-ACPD), stimulated the release of [3H]dopamine, in a dose-dependent manner. When magnesium was omitted or in the presence of AMPA, NMDA also increased the release of [3H]dopamine. The effects of AMPA and kainate were competitively inhibited by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) or 6,7-dinitro-quinoxaline-2,3-dione (DNQX), whereas those of NMDA were reduced by 2-amino-5-phosphonovalerate (APV) or (+)-5-methyl-10,11-dihydro-5-H-dibenzo(a,d)cyclo-hepten-5,10-imine maleate (MK801). The stimulation of [3H]dopamine release by a high concentration of glutamate resulted from the concomitant activation of AMPA and NMDA receptors since this effect was potentiated by glycine and reduced by 2-amino-5-phosphonovalerate or MK801. This reduction was almost complete in the combined presence of DNQX and MK801. Surprisingly, glutamate and NMDA (in the absence of magnesium) reduced the efflux of [3H]water. The reduction of [3H]dopamine synthesis was blocked by 2-amino-5-phosphonovalerate indicating the involvement of NMDA receptors. Neither AMPA nor kainate affected dopamine synthesis. The inhibition of [3H]dopamine synthesis resulting from the stimulation of NMDA receptors was prevented when synaptosomes were continuously superfused with adenosine deaminase and quinpirole, a combined treatment known to markedly reduce the phosphorylation of tyrosine hydroxylase by cAMP-dependent protein kinase. The opposite effects of a high concentration of glutamate on [3H]dopamine synthesis and release were mimicked by ionomycin. As a working hypothesis, it is proposed that the NMDA-triggered calcium influx could lead to a reduction of tyrosine hydroxylase phosphorylation, possibly through an activation of calcineurin.