Dopaminergic systems have been known to be involved in the regulation of locomotor activity and development of psychosis. However, the observations that some Parkinson's disease patients can move effectively under appropriate conditions despite low dopamine levels (eg, kinesia paradoxia) and that several psychotic symptoms are typical antipsychotic resistant and atypical antipsychotic sensitive indicate that other systems beyond the dopaminergic system may also affect locomotor activity and psychosis. The present study showed that dopamine-deficient (DD) mice, which had received daily L-DOPA injections, could move effectively and even be hyperactive 72 h after the last L-DOPA injection when dopamine was almost completely depleted. Such hyperactivity was ameliorated by clozapine but not haloperidol or ziprasidone. Among multiple actions of clozapine, muscarinic acetylcholine (ACh) activation markedly reduced locomotor activity in DD mice. Furthermore, the expression of choline acetyltransferase, an ACh synthase, was reduced and extracellular ACh levels were significantly reduced in DD mice. These results suggest that the cholinergic system, in addition to the dopaminergic system, may be involved in motor control, including hyperactivity and psychosis. The present findings provide additional evidence that the cholinergic system may be targeted for the treatment of Parkinson's disease and psychosis.