A prolongation of the ventricular effective refractory period in response to cholinergic agonists or vagal stimulation has been demonstrated in a number of in vivo animal models. However, exposure of isolated myocardial tissues obtained from these hearts to as much as 10-4 M acetylcholine has been shown to produce essentially no change in action potential duration or effective refractory period. The discrepancy between the in vivo and in vitro findings generally has been explained on the basis of accentuated antagonism, whereby parasympathetic agonists exert their influence through antagonism of the effects of β-adrenergic tone in vivo. The fact that acetylcholine exerts little if any direct effect on the electrical activity of ventricular myocardium, although well accepted, is based exclusively on studies performed using endocardial preparations. Our recent demonstration of major electrophysiological differences between canine ventricular endocardium and epicardium prompted us to examine the effects of acetylcholine and the role of accentuated antagonism in these two tissue types. Using standard microelectrode techniques, we show that acetylcholine (10-7 - 10-5 M) has little if any effect in canine ventricular endocardium but a pronounced effect to either prolong or markedly abbreviate action potential duration and effective refractory period in epicardium. These effects of acetylcholine on epicardium are attended by an accentuation of the spike and dome morphology of the action potential, are readily reversed with atropine, fail to appear when epicardium is pretreated with the transient outward current blocker 4-aminopyridine, are accentuated in the presence of isoproterenol (10-7 to 5 x 10-6 M), and persist in the presence of propranolol. Isoproterenol-induced abbreviation of action potential duration and effective refractory period is also shown to be more pronounced in epicardium than in endocardium; equimolar concentrations of acetylcholine completely antagonize the effects of isoproterenol in endocardium and epicardium. We conclude that acetylcholine exerts important direct effects on the electrical response of canine ventricular myocardium, which are accentuated in the presence of β-adrenergic agonists. Our findings suggest that differential response of epicardium and endocardium to acetylcholine is due to the presence of a transient outward current-mediated spike and dome morphology in the epicardial action potential. Finally, the data suggest that acetylcholine may exert antiarrhythmic as well as arrhythmogenic effects through its actions to alter conduction and refractoriness.