Abstract3,4‐Methylenedioxymethamphetamine (MDMA), an amphetamine analog, has been shown recently to increase the release of acetylcholine (ACh) in the prefrontal cortex (PFC). The present study further characterizes the stimulatory effect of MDMA on cortical ACh release and examines the role of serotonin (5‐HT) and dopamine (DA) receptors in this response. The extracellular concentration of ACh was increased dose‐dependently and similarly by the (+) and (−) enantiomers of MDMA (5 and 20 mg/kg, i.p.). The systemic administration of the 5‐HT4antagonist SDZ 205,557 (1 mg/kg, i.p.), but not the 5‐HT2A/2B/2Cantagonist LY‐53,857 (3 mg/kg, i.p.), significantly decreased cortical ACh release induced by MDMA. The MDMA‐induced increase in the extracellular concentration of ACh also was significantly blunted in rats treated with the D1receptor antagonist SCH 23390 (0.5 mg/kg, i.p.). The extent to which the coadministration of SDZ 205,557 and SCH 23390 suppressed the MDMA‐induced release of ACh in the PFC was no greater than that produced by either antagonist alone. These results suggest that the 5‐HT4and D1receptor subtypes contribute to the mechanism by which MDMA increases ACh release in the PFC. Synapse 58:229–235, 2005. © 2005 Wiley‐Liss, Inc.