The atrioventricular (AV) ring tissue (surrounding the atrial orifices of tricuspid and mitral valves) and the right ventricular outflow tract (RVOT) are major sources of ectopic electrical activity causing arrhythmias (1, 2). The aim of this study was to characterise these tissues in structurally normal hearts, identify whether they are predisposed to generate arrhythmias and investigate the mechanisms underlying arrhythmogenicity. Male Wistar rats (275-375 g) were killed in accordance with the United Kingdom Animals (Scientific Procedures) Act, 1986. Isolated tissue preparations were superfused at 37°C and extracellular electrograms were recorded with a multi-electrode array and intracellular action potentials (APs) were recorded using microelectrodes. Ion channel expression was characterised using qPCR and immunohistochemistry. Data are presented as means±SEMs and ‘n’ denotes number of animals. In right atrial preparations, multi-electrode array recordings showed the leading pacemaker site at the level of the sinus node; AV ring APs were atrial-like (n=2), showing a maximum diastolic potential (MDP) of -82±1 mV, a fast upstroke velocity (dV/dtmax, 205±17 V/s), a large amplitude (102±2 mV) and the time to 50% (T50) and 90% (T90) repolarization of 22±1 ms and 80±1 ms, respectively. Upon detaching the sinus node, spontaneous activity originated in the AV ring and propagated towards the terminal crest. Intracellular APs recorded at the leading pacemaker site in the AV ring were sinus node-like. Spontaneous APs occurred at a frequency of 2.1±0.3 Hz (n=4) and were characterised by a MDP of -50±3 mV, a slow upstroke (dV/dtmax, 7±1 V/s), a small amplitude (49±3 mV) and slow diastolic depolarization. The T50 and T90 were 46±5 ms and 87±8 ms, respectively. Pacemaking in the AV ring was accelerated by treatment with the β-adrenergic agonist isoproterenol (0.05 µM) and could be inhibited by either blocking the pacemaker current If with 2 mM Cs+ or disrupting Ca2+ release from the sarcoplasmic reticulum with ryanodine (2 μM). In RVOT preparations, spontaneous electrical activity was observed in 2 out of 7 cases. The leading pacemaker site was located immediately inferior to the pulmonary valve and again showed sinus node-like APs (MDP, -41±2 mV; dV/dtmax, 25±7 V/s; amplitude, 44±12 mV; n=2) with prominent diastolic depolarization. Curiously, both the right AV ring and the RVOT showed bursting activity. Consistent with these observations, qPCR and immunolabelling showed an expression pattern of ion channels, Ca2+-handling proteins and receptors in the right AV ring and RVOT similar to that in the sinus node. The AV rings and the RVOT of the normal heart are capable of pacemaker activity and this is the likely basis of arrhythmias originating in these tissues.