We studied the role of the anticholinergic properties of disopyramide and quinidine in mediating the electrophysiological effects of these agents on isolated cardiac tissue. In right atria, disopyramide, quinidine, and procainamide administered alone elicited negative chronotropic responses. However, after cholinergic stimulation with physostigmine (1 × 10"6M), disopyramide and quinidine produced a positive chronotropic response. Procainamide, when administered under the same conditions of increased cholinergic stimulation, elicited negative chronotropic responses similar to those observed when it was given alone. In Purkinje fibers, disopyramide, quinidine, and procainamide superfused alone for 5 minutes significantly prolonged action potential duration (APD). When administered to Purkinje fibers pretreated with isoproterenol (1 × 10−7M) plus acetylcholine (3 × 10−7M), disopyramide and quinidine shortened APD. Atropine also shortened APD of fibers exposed to both isoproterenol and acetylcholine. In these experiments, isoproterenol consistently shortened APD, and acetylcholine consistently blunted these effects of isoproterenol. Thus, disopyramide and quinidine, alone, prolong APD, but when given in the setting of increased adrenergic-cholinergic stimulation, they shorten APD. Procainamide, when administered to Purkinje fibers pretreated with isoproterenol and acetylcholine, prolongs APD as when it is administered alone. These data demonstrate that disopyramide and quinidine exert important anticholinergic electrophysiologic effects on the atria and the ventricular conducting systems. These anticholinergic effects may contribute to the antiarrhythmic and arrhythmogenic properties of these compounds. Circ Res 46: 660-668, 1980