To clarify the findings that clozapine is both a muscarinic receptor agonist and antagonist, we examined the effects of neuroleptics on forskolin-stimulated cAMP accumulation in Chinese hamster ovary cells expressing human muscarinic m4 receptors (CHO-hm4) and in rat striatum. With CHO-hm4 cells, clozapine induced a concentration-dependent and atropine-sensitive inhibition on cAMP formation, with EC50 = 60 nM and Emax = 74% of carbachol maximum. Other atypical neuroleptics, fluperlapine, tenilapine and olanzapine, were similar but less potent, while risperidone, rilapine, quetiapine (ICI 204,636), sertindole, and ziprasidone had almost no effect. Typical neuroleptics, haloperidol, chlorpromazine, fluphenazine, thiothixene, thioridazine, and molindone, showed either no effect or an atropine-resistant inhibition of cAMP formation. However, in rat striatal tissues, clozapine, up to 10 microM, did not show a significant inhibition of cAMP formation, probably due to a relatively low abundance of muscarinic m4 receptors and the presence of multiple types of muscarinic and other receptors, with which clozapine interacts. Nevertheless, muscarinic m4 receptor agonism, to some extent, may be a relevant mechanism for the therapeutic efficacy and side effects of clozapine and some atypical neuroleptics.