The pharmacological basis of acute (±)-MDMA (3,4-methylenedioxymethamphetamine) intoxication still awaits full characterization. According to present knowledge, MDMA enhances the release of serotonin and dopamine in striatal slices and interacts with different types of receptors such as 5-HT2 (5-hydroxytryptamine or serotonin), M1 and M2 muscarinic acetylcholine (ACh), and histamine H1 receptors. Currently, no information is available about the influence of (±)-MDMA on striatal cholinergic neurotransmission. In the present study, we used the in vitro perfusion technique to investigate the effect of (±)-MDMA on ACh release in rat striatal slices. Perfusions with (±)-MDMA (10–300 μm) resulted in a dose-dependent increase of spontaneous ACh release (EC50≈ 30 μm). The effect was reversible and Ca++- and tetrodotoxin-sensitive. To determine the neurochemical pathways underlying this response, we perfused with (±)-MDMA in the presence of various inhibitors of neurotransmitter receptors. Blockade of glutamate or muscarinic ACh receptors as well as 5-HT1, 5-HT2, 5-HT3C or dopamine D2 receptors did not modulate (±)-MDMA–induced ACh release. However, the presence of histamine H1 receptor antagonists in the perfusion medium abolished (±)-MDMA-induced ACh release. The present data clearly demonstrate that (±)-MDMA enhances the activity of striatal cholinergic neurons and suggest an involvement of histamine H1 receptors. The effect is not mediated by glutamate and does not involve the activation of receptors of dopamine D2, 5-HT1, 5-HT2, 5-HT3C or muscarinic ACh. Considering the relatively high affinity of (±)-MDMA for the H1 histamine receptor (Ki 6 μm), a direct activation of this type of receptor might represent a plausible mechanism for (±)-MDMA-induced ACh release.