The possible interaction between the muscarinic receptor and the antiarrythmic drug amiodarone was studied physiologically in the guinea pig ileum, as well as by competition binding experiments in rat brain and cardiac tissues, using the highly specific tritiated muscarinic antagonist N-methyl-4-piperidyl benzilate. In these studies, amiodarone was found to affect both antagonist and agonist binding to the muscarinic receptor. The drug's inhibitory effect on the binding of antagonist to cerebral cortex muscarinic receptors was consistent with mutually exclusive binding of the compounds [KI = (1.0 +/- 0.2)10(-5) M]. On the other hand, in the brain stem and in cardiac tissues (atrium and ventricle) the inhibitory effect on the binding of muscarinic antagonist could not be fitted to a simple model of competitive inhibition. The possible mode of interaction is discussed. Compared with its activity in the cerebral cortex, amiodarone was a more potent inhibitor of muscarinic antagonist binding in the brain stem and in the atrium and ventricle of the heart [apparent KI values were (6.5 +/- 0.1)10(-6), (4.0 +/- 0.1)10(-6), and (4.0 +/- 0.1)10(-6) M, respectively]. In view of the KI values and the serum concentration of amiodarone observed therapeutically (10(-6) M), the effect of amiodarone on the muscarinic system may have clinical relevance. In both the brain stem and the cardiac preparations, amiodarone converted sites that bind agonist with high affinity into low-affinity sites. Agonist binding in the cerebral cortex was not affected.