In the cerebellum there is scant evidence for cholinergic transmission but a large amount of acetylcholinesterase. Although it exists most commonly in a membrane-bound form, the release of a soluble form of this enzyme, within the cerebrum, has indicated that it may have a novel non-cholinergic role. In order to understand why the cerebellum is rich in acetylcholinesterase, the first step has been to investigate the possibility of its release in this structure. Following unilateral application of a depolarizing concentration of potassium ions, there was a large, sustained, calcium-dependent increase in release of acetylcholinesterase, specifically in the local cerebellar cortex; a marked enhancement of acetylcholinesterase release also occurred in the contralateral cerebellum, suggesting that the phenomenon reflected polysynaptic neuronal events. Indeed, systemic administration of harmaline, which modifies activity in certain cerebellar afferent pathways, induced a significant increase in acetylcholinesterase release in the cerebellar cortex. Local administration of the cholinomimetic, carbachol, had no effect. It is concluded that acetylcholinesterase is released from cerebellar neurons in association with physiological events, yet unrelated to cholinergic transmission.