Stimulus-evoked dopamine overflow in rat neostriatal slices was determined using fast cyclic voltammetry. The dopamine efflux induced by intrastriatal stimulation increased with stimulus intensity and was found to be enhanced by more than 100% upon application of the dopamine uptake inhibitor nomifensine. The acetylcholine esterase inhibitor eserine concentration-dependently and reversibly depressed stimulus-induced dopamine overflow. This effect was mediated by both, muscarinic and nicotinic cholinergic receptors: the action of eserine was mimicked by cholinergic agonists (muscarine and nicotine) and the effects of these agonists were blocked by muscarinic and nicotinic antagonists (atropine and dihydro-β-erythroidine). These experiments suggest that endogenous acetylcholine exerts an inhibitory control on stimulus-evoked (i.e. phasic) dopamine overflow in vitro by affecting striatal dopaminergic nerve terminals.