An unexplained action of acetylcholinesterase (AChE) has previously been demonstrated on motor behaviour in the substantia nigra: a single infusion of the protein induced long-term circling behaviour, reflecting sustained increased activity of the nigro-striatal pathway. In this study, a highly purified form of AChE was infused and the long-term behavioural effects were still observed. In addition, recombinant human AChE produced a similar response, suggesting that AChE itself, and not a contaminant, was responsible for these behavioural actions. Butyrylcholinesterase, which also hydrolyses acetylcholine, was without effect. Hence AChE is not exerting these actions by potentiating the neurotransmitter acetylcholine, but via some cholinergic-independent mechanism.