Within the substantia nigra acetylcholinesterase (AChE) has a novel non-cholinergic action that is functionally manifest as chronic circling behaviour in rats. The aim of this study was to explore the possible biochemical mechanisms that could underlie the long-term behavioural effects of this protein, infused unilaterally into one substantia nigra. A single treatment of acetylcholinesterase induced modest but consistent circling behaviour in the presence of a systemic amphetamine challenge for the maximum time tested, up to 50 days: comparable infusions of saline were without effect. When animals received a challenge of the direct dopamine agonist apomorphine, no AChE-induced circling was observed: this result suggested that the phenomenon did not entail a down-regulation of striatal dopamine receptors. On the other hand, a challenge of the dopamine uptake inhibitor nomifensine resulted in AChE-induced circling that was indistinguishable from that seen in the presence of amphetamine: hence the circling behaviour seen could be attributable to an AChE-induced increase in availability of extracellular dopamine. In animals where AChE caused contraversive rotation, indicative of an enhanced activity in the nigrostriatal pathway, there was a significant elevation in the dopamine content of the striatum on the treated side. It is concluded that AChE can chronically enhance the release of dopamine from the nigrostriatal pathway such that motor behaviour is correspondingly modified, but to an extent sufficiently modest to avoid compensatory synaptic reversal mechanisms.