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Sleep and arousal are both important for animals. The neurotransmitter acetylcholine (ACh) has long been found to promote both sleep and arousal in mammals, an apparent paradox which has also been found to exist in flies, causing much confusion in understanding sleep and arousal. Here we have systematically studied all 13 ACh receptors (AChRs) in Drosophila to understand mechanisms underlying ACh function in sleep and arousal. We found that exogenous stimuli-induced arousal was decreased in nAChRα3 mutants, whereas sleep was decreased in