The actions of excitatory amino acids on the release of previously incorporated gamma-[3H]aminobutyric acid ([3H]GABA) were examined in purified (greater than 93%) striatal neurons derived from the fetal mouse brain and differentiated in primary culture. Glutamate, KCl, and veratrine evoked a dose-dependent, saturable, and reversible release of [3H]GABA from striatal neurons. Glutamate actions were not reduced in the absence of calcium, and were insensitive to tetrodotoxin. The dose-response relationships of excitatory amino acids demonstrated the following rank order of potency: glutamate greater than aspartate = N-methyl-D-aspartate greater than kainate much greater than quisqualate. Kainate, however, was the most effective agonist, evoking an eightfold increase over baseline levels of [3H]GABA release. Aspartate- and N-methyl-D-aspartate-evoked release was abolished in the presence of either 2-aminophosphonovaleric acid or gamma-D-glutamylglycine. Release due to glutamate and kainate was partially or ineffectively attenuated by these agents. Glutamate-, aspartate-, and N-methyl-D-aspartate-evoked GABA releases were augmented when calcium was omitted from the bathing medium and reduced when sodium was replaced with choline or lithium. Kainate-evoked release was unaffected when calcium was omitted, virtually unchanged when choline replaced sodium, and markedly potentiated when lithium was substituted for sodium. These findings suggest that at least two distinct receptor systems for excitatory amino acids mediate the evoked release of [3H]GABA from striatal neurons in primary culture. These two systems, aspartate/N-methyl-D-aspartate- and kainate-preferring, are distinguishable on the basis of their pharmacological and ionic properties.