Cardiopulmonary bypass (CPB) is associated with an ischemia-reperfusion injury which can potentially impact the rhythmicity of the heart. Junctional Ectopic Tachycardia (JET) is the most common post-operative arrhythmia seen in neonates and infants undergoing CPB for repair of heart defects. Clinical evidence suggests JET originates from the AV node area and ischemia is speculated to be a pathogenic factor. JET is also associated with the post-operative administration of inotropes, in particular dopamine. To determine how ischemia-reperfusion affects the pacemaker cell activity in neonates, AV nodal (AVNC) and SA nodal cells (SANC) were isolated from 10-day old New Zealand white rabbits and spontaneous action potentials were recorded using the perforated current clamp technique. Rabbits were anesthetized with thiopental sodium (60mg/kg) through intra-peritoneal injection prior to thoracotomy. The heart was rapidly excised and perfused with collagenase and subsequently protease in the Langendorff mode. AVNCs were dissociated from the AVN tissue cut at the triangle of Koch. SANCs were dissociated from the SAN tissue located adjacent to the crista terminalis. AVNCs and SANCs were identified by their automaticity, input resistance, and pacemaker current expression. We recorded APs during pre-ischemia, ischemia, post-ischemia with drugs and reperfusion with normal Tyrode’s solution. After 5 min exposure to the ischemia solution (pH 7.0, 5 mM lactate, 0 mM glucose), action potential (AP) frequency on AVNCs decreased and the AP duration was shortened (p<0.05, n=8, paired-samples t test); ischemia had no significant effect on SANCs over the same time period. Our results showed 20 μM dopamine consistently led to irreversible irregular APs during reperfusion on AVNCs (n=4) but not on SANCs. Typical changes in AP pattern were: slower upstroke, unstable maximal diastolic potentials, and an increase in early and delayed afterdepolarizations (EAD and DAD). Isoproterenol (1 μM) also induced AP changes in AVNCs (n=3) during reperfusion but to a lesser extent, without EAD but with DAD. We conclude AVNCs are more susceptible to ischemia than SANCs in neonates; we also demonstrate that the post-ischemia application of β-adrenergic agonists may exacerbate the trauma from ischemia and lead to irregular electrical activity in AVNCs during reperfusion. Whether JET results from a deterioration of AV conduction or abnormally enhanced AV automaticity needs to be explored further.