Flecainide can be an effective treatment for catecholaminergic polymorphic tachycardia (CPVT) in both animal models and humans [1]. While flecainide is an inhibitor of Nav1.5, initial studies on adult ventricular myocytes (AVMs) from wild type (WT) rat or CPVT (calsequestrin-2 knockout) mouse concluded that flecainide acts primarily on RyR2 to reduce the frequency of spontaneous Ca2+ waves (SCWs) [1, 2]. However, in subsequent studies it was suggested (i) that the antiarrhythmic effect of flecainide was due to an action on Nav1.5, not RyR2 in WT rat AVMs [3, 4] and (ii) that flecainide does not affect gating of isolated RyR2 under physiologically relevant conditions [4]. Here, the effects of flecainide were further investigated in permeabilised Wistar rat AVMs using confocal imaging [2]. Data were analysed using one or two way repeated measures ANOVA where appropriate, and are presented as mean ± SEM. Flecainide (25 µM) decreased SCW frequency by 21.2 ± 5.43 % (n=12; p<0.01). This compares with previous findings on permeabilized AVMs from CPVT mice, where a lower level of flecainide (6 µM) caused a 50 % greater decrease in wave frequency [5]. Interestingly however, when the SR counter-current was inhibited by substitution of K+ with Cs+, flecainide (25 μM) reduced SCW frequency by 42.8 ± 13.6 % (n=13; p<0.001) in WT rat AVMs. This might be explained if a transient polarisation of the SR membrane during SR Ca2+ release (inside negative) facilitates the action of flecainide on RyR2. In further experiments, flecainide entry into intact WT rat AVMs was detected using a fluorescent (FITC) labelled form (flecainide-F). When 6 μM flecainide-F was added to the extracellular solution, the intracellular fluorescence (IF) increased progressively and after 30-40 min, IF approached the extracellular fluorescence (EF). After 3 hours in 6 μM flecainide-F, the IF was 2.32 ± 0.17 times the EF (n=11-12; p<0.0001), suggesting accumulation of flecainide-F. These data show that flecainide has qualitatively similar effects on SCWs in WT rat to those reported in CPVT mouse AVMs [2], although a higher intracellular flecainide concentration was required in WT rat AVMs (25 µM vs 6 μM) and the decrease in wave frequency was less pronounced. The flecainide-F experiments suggest that it may take several hours incubation to achieve the intracellular drug level needed to affect RyR2 in WT rat AVMs. The greater effect of flecainide following counter-current inhibition may relate to recent findings on isolated RyRs within lipid bilayers, where flecainide preferentially blocked the channel when there was a negative charge across the bilayer.