Flecainide is effective at preventing delayed after depolarisations and associated arrhythmias in catecholaminergic polymorphic ventricular tachycardia (CPVT), however its primary mode of action remains contentious (1-4). While initial studies using CPVT transgenic mice concluded that flecainide had a direct action on the ryanodine receptor (RyR2), which reduced Ca2+ wave frequency (1), subsequent reports suggested that its effects are entirely due to Na+ channel (Nav1.5) block (2-4). In this study, the effects of flecainide on sarcoplasmic reticulum Ca2+ regulation were investigated in saponin permeabilised ventricular myocytes isolated from adult male Wistar rats. Rats were killed according to Schedule 1 methods. Following isolation and permeabilisation, cells were perfused with a mock intracellular solution containing fluo-3 pentapotassium salt (15 µM) and Ca2+ release events were visualised using line scan confocal microscopy. Ca2+ sparks were automatically detected and analysed using the Sparkmaster plugin for ImageJ. Statistical significance (p<0.05) was determined using a paired Student’s t-Test or one way repeated measures ANOVA. Values are mean ± SEM, n=cell number. Ca2+ wave frequency was investigated after 3 min perfusion with or without 25 µM flecainide and results were grouped according to initial wave frequency. At low wave frequencies (<5 waves/min or 6-9 waves/min), 25 µM flecainide significantly reduced wave frequency by 25.8 ± 7.8 % (p<0.01, n=15) and 14.6 ± 4.6 % (p<0.01, n=17) respectively. At high initial wave frequencies (>10 waves/min) there was no significant change (p>0.05, n=14). There was no significant change in wave frequency in time-dependent controls within any group (p>0.05, n=7). In separate experiments, the effects of flecainide on Ca2+ sparks were studied in the absence of Ca2+ waves. The amplitude of Ca2+ sparks decreased by 15.5 ± 2.97 %, (p<0.001, n=30); width significantly decreased by 15.9 ± 1.66 % (p<0.001, n=30); frequency significantly increased by 36.0 ± 10.4 % (p<0.01, n=30); and there was no significant change in spark duration (p>0.05, n=30). There was no significant change in spark properties in time-dependent controls (n=10, p>0.05). These experiments provide evidence that in wild-type rat myocytes, flecainide can produce qualitatively similar changes in Ca2+ sparks and waves to those reported in permeabilised myocytes from CPVT mice, albeit at a higher drug concentration (25 vs 6 µM) (1). The data are consistent with an effect on RyR2 gating under conditions where the channel remains in situ and the influence of Nav1.5 is excluded. The apparent ineffectiveness of flecainide at high initial Ca2+ wave frequencies may contribute to recent discrepancies in the literature.