The class 1c anti-arrhythmic flecainide is a well-characterized use-dependent blocker of the cardiac Na+ channel. Elucidation of the mechanisms involved in this block has been facilitated by the use of fully charged (QX-FL) and neutral (NU-FL) derivatives of flecainide 1. In these studies it was established that Na+ channel block requires entry of the cationic form of the molecule into the cytosolic vestibule of the open channel. Flecainide has recently been shown to be effective in the treatment of catecholaminergic polymorphic ventricular tachycardia (CPVT), however the mechanism of flecainide action is contentious 2-4. In this study, we have sought to determine how the flecainide derivatives influence RyR2-mediated Ca2+-release from the sarcoplasmic reticulum and whether this correlates with their effectiveness as Na+ channel blockers and/or blockers of RyR2. To investigate this, we compared the ability of flecainide, QX-FL and NU-FL to modulate the properties of Ca2+ sparks in intact adult rat cardiac myocytes with their ability to block cation flux in individual recombinant human RyR2 channels reconstituted into planar lipid bilayers. NU-FL was synthesized by a novel route. Cardiomyocytes were dialyzed via patch pipette with either vehicle control solution, or with flecainide, QX-FL or NU-FL (5μM) and were stimulated via depolarization from -80 to +30 mV (2Hz, 30s). Spontaneous Ca2+ sparks were recorded during a subsequent 20 second quiescent period. Data are given as mean±SEM. Intracellular application of flecainide or QX-FL resulted in equivalent suppression of Ca2+ spark frequency (34.96 ± 6.23% and 28.49 ± 9.86% reduction, respectively, p<0.05 (unpaired t-test) compared to control cells) whereas NU-FL did not reduce Ca2+ spark frequency. Flecainide, QX-FL and NU-FL had no measurable effect on Ca2+ spark amplitude or mass. For single channel studies, we reconstituted recombinant human RyR2 channels into planar lipid bilayers under voltage clamp conditions in symmetrical 610mM KCl. We found that both QX-FL and NU-FL were dose- and voltage-dependent open channel blockers of the non-physiological cytosolic to luminal flux of ions through the channel but were significantly less effective than flecainide (n=6). The fractional conductances of the blocked state were different for each compound. However, neither cytosolic QX-FL nor NU-FL was able to influence the luminal to cytosol flux of cations through the RyR2 channel. Given its inability to block this physiologically-relevant cation flux through RyR2, the effect of QX-FL on Ca2+ sparks is likely, by analogy with flecainide, to result from block of the Na+ channel. Our data reveal important information about differences in the nature of the interaction of flecainide with sites in the cytosolic vestibules of Na+ and RyR2 channels.