Objective · To study the electrophysiological effect of (S)-OTS·HCl on the heart. Methods · The conventional intracellular recording, electrocardiograph (ECG) and Langendorff cardiac perfusion technique were employed to investigate the effect of (S)-OTS·HCl on in-vivo and in-vitro hearts of guinea pigs and rabbits. Results · (S)-OTS·HCl could bind to M2 muscarinic receptors and dose-dependently prolong the RR intervals significantly in vivo. It had no effect on resting potential (RP), action potential amplitude (APA), and maximum upstroke velocity of phase 0 (Vmax) of ventricular myocytes. Instead, 1×10-5 mol/L (S)-OTS·HCl could shorten the action potential duration at 50 percent repolarization (APD50) and APD90 to 91.6% and 90.9%, respectively. And the spontaneous depolarization rate of phase 4 (SDR) of sinus nodes was reduced to its 13.7% when rabbit sinus nodes were exposed to 1×10-7 mol/L (S)-OTS·HCl. (S)-OTS·HCl could inhibit Ca2+channel effectively. It decreased APA and Vmax of sinus nodes and attenuated the cardiac contractility in vitro. Conclusion · (S)-OTS·HCl is a potent cholinergic agonist and has negative chronotropic, dromotropic, and inotropic effects on hearts via binding to M2 muscarinic receptors.