The effect of metabotropic glutamate receptor agonists and antagonists on KCl (20 mm)-induced endogenous acetylcholine release from rat striatal synaptosomes was investigated. The group I agonist (S)-3,5-dihydroxyphenylglycine (DHPG), 1–1000 nm, potentiated in a concentration-dependent way the KCl-induced acetylcholine release, reaching maximal efficacy at 100 nm (+93 ± 14%). The effect of DHPG (10 nm) was counteracted by coapplication of (7-hydroximino)cyclopropan-b-chromen-1a-carboxylate (CPCCOEt), 10 µm, a metabotropic glutamate receptor type one selective antagonist, and 2-methyl-6-(phenylethynyl)pyridine (MPEP), 10 µm, a metabotropic glutamate receptor type five selective antagonist, but not by application of either antagonist alone. The group II agonist (2S, 1′R, 2′R, 3′R)-2-(2,3-dicarboxycyclopropyl)glycine (DCG-IV), 1–1000 nm, inhibited in a concentration-dependent way the KCl-induced acetylcholine release displaying maximal efficacy at 300 nm (−32 ± 2%). The effect of DCG-IV 300 nm was counteracted by the group II selective antagonist (2S)-α-ethylglutamic acid (EGLU), 300 µm. The group III agonist l-amino-4-phosphonobutyric acid (L-AP4) failed to alter the KCl-induced acetycholine release up to 300 µm. We conclude that metabotropic glutamate receptors belonging to group I and II are located on the axon terminals of striatal cholinergic interneurons, their activation resulting in facilitation and inhibition, respectively, of acetylcholine release.