It has previously been shown that neuroleptic drugs block an apparently inhibitory influence of dopamine on cholinergic interneurons in striatum, thereby increasing acetylcholine turnover. In this study, systemic administration of the neuroleptic, fluphenazine, decreased the acetylcholine content in the striatum but not the neocortex of rats killed by focussed microwave irradiation. The effect was observed with doses of fluphenazine as low as 0.05 mg/kg, and was also seen after two other neuroleptics, spiroperidol (1 mg/kg) and haloperidol (4 mg/kg). In contrast, neither fluphenazine nor haloperidol pretreatment had any effect on the high affinity accumulation of choline by striatal synaptosomes. These observations suggest that after administration of dopamine receptor antagonists the release and metabolism of acetylcholine in the striatum is increased, but that a compensatory increase in choline uptake does not occur, thereby resulting in a temporary decrease in acetylcholine concentration. On the basis of these findings, we conclude that acetylcholine synthesis is regulated differently in the striatum than in other brain regions.