Brain microdialysis coupled to high performance liquid chromatography with electrochemical detection (HPLC–ED) was used to evaluate the influence of melatonin on extracellular concentration of acetylcholine (ACh) in the nucleus accumbens (NAc) of rats. Motor activity was simultaneously monitored during the dialysis sessions with an activity meter. Melatonin and prazosin were administered locally through the dialysis probe. It was found that melatonin dose-dependently increased accumbens ACh. Melatonin (3 μM) decreased horizontal activity and increased vertical activity, while another dose (100 μM) enhanced both horizontal and vertical activity. Prazosin, a putative melatonin antagonist, blocked the effects of melatonin on both motor activity and ACh release when given 20 min before melatonin. Overall, these results suggest that melatonin modulates the release of ACh in the NAc and the pattern of motor activity in the rat.