We have investigated the actions of acetylcholine in the absence and presence of the beta-agonist isoproterenol in cardiac Purkinje fibers. beta-Agonists, like isoproterenol, increase the magnitude of the pacemaker current (If) in cardiac myocytes by shifting its activation voltage more positive on the voltage axis. We find that acetylcholine has no effect on If in the absence of isoproterenol. However, if If is first increased by beta-agonist stimulation, acetylcholine can then return If to control levels. This effect on If is exerted through muscarinic receptors since atropine prevents this action of acetylcholine. Functionally, this action of acetylcholine can guarantee the maintenance of ventricular pacemakers when there is high parasympathetic tone but can also prevent extra ventricular beats when sympathetic and parasympathetic tone are both high.