The pharmacological nature of the interaction of excitatory amino acids with striatal cholinergic neurons was investigated in vitro. Agonists of excitatory amino acid receptors evoked the release of [3H]acetylcholine from slices of rat striatum, in the presence of magnesium (1.2 mM). Removal of magnesium from the medium markedly increased the release of [3H]acetylcholine evoked by all excitatory amino acid receptor agonists tested, with the exception of kainate. In the absence but not the presence of magnesium, a clear rank order of potency was found: N-methyl-DL-aspartate = ibotenate greater than L-glutamate greater than L-aspartate greater than or equal to cysteate greater than kainate = quisqualate. The excitatory amino acid receptor mediating [3H]acetylcholine release resembles the N-methyl-D-aspartate preferring (N-type) receptor, as previously characterized electrophysiologically, according to 3 criteria: (1) rank order of potency of agonists; (2) magnesium-sensitivity; and (3) antagonism by 2-amino-5-phosphonovalerate. The release of [3H]acetylcholine evoked by N-methyl-DL-aspartate was blocked by tetrodotoxin (0.5 microM). Moreover, N-methyl-DL-aspartate failed to evoke [3H]acetylcholine release from slices of hippocampus, where cholinergic afferents, rather than interneurons, are found. These results suggest that excitatory amino acids act at receptors on the dendrites of striatal cholinergic interneurons, giving rise to action potentials and release of acetylcholine from cholinergic nerve terminals.