Abstract The effects of various neuroleptics and of apomorphine on the metabolism of ACh were examined in the neostriatum of the rat. For this purpose, a specific radio-enzymatic assay for brain ACh was used. This method is based on the preliminary purification of the choline esters by liquid cation exchange, separation of choline and ACh on thin layer chromatography plates, hydrolysis of ACh then reacetylation of the choline moiety with a purified and stabilized rat brain choline acetyltransferase. The rat neostriatal ACh levels were decreased by neuroleptics of the phenothiazine and butyrophenone type and increased by apomorphine. An ‘ in vivo’ estimation of the rate of utilization of ACh was obtained by measuring the decline in neostriatal ACh content following the local microinjection of hemicholinium-3. This compound blocked almost totally the synthesis of ACh in these conditions. Chlorpromazine (15 mg/kg) enhanced neo-striatal ACh utilization and apomorphine (10 mg/kg) antagonized this effect. Neuroleptics did not affect ACh levels in the parietal cerebral cortex and the hippocampal formation. The modifications of the activity of neostriatal cholinergic neurons by chlorpromazine and apomorphine were still observed following the degeneration of the nigro-neostriatal dopaminergic fibers induced by the injection of 6-hydroxydopamine into the substantia nigra. The results strongly suggest that dopaminergic receptors as defined by their pharmacological interaction with neuroleptics and apomorphine are localized on neostriatal ACh neurons.