Previous studies have suggested that melatonin, released from photoreceptors, may modulate retinal dark-adaptive responses by inhibition of dopamine release from retinal interneurons. We have broadened these studies to examine the effect of melatonin on release of another retinal neurotransmitter, acetylcholine (ACh). The ACh system in rabbit retina has been localized to starburst amacrine cells, which release ACh in response to a variety of experimental stimuli, including direct potassium depolarization, flashing light, and glutamatergic as well as GABAergic inputs. The effect of melatonin on release of endogenously synthesized [3H]-ACh was measured in perfusates from retinas or retinal synaptosomes preloaded with [3H]-choline chloride. Melatonin significantly inhibited ACh release stimulated by potassium in intact retina but not in synaptosomes. Stimulation of intact retina by flashing light or by the glutamate receptor agonist, kainic acid, was also inhibited by melatonin. In contrast, there was no significant effect of melatonin on picrotoxin-induced release. These findings suggest that melatonin does have an inhibitory effect on ACh release, either by direct interaction with the cholinergic amacrine cell, or indirectly via GABAergic but not glutamatergic neurons.