Flecainide is a Class Ic (Na+ channel blocking) antiarrhythmic drug widely use in clinical treatment of atrial fibrillation that has been associated with an increased risk of arrhythmic events in post-myocardial infarction patients (Echt et al., 1991). In addition to its Na+ channel blocking activity, flecainide can inhibit hERG K+ channels at clinically relevant concentrations (Paul et al., 2002; Du et al., 2011; Breindahl et al., 2000). Flecainide’s potency against hERG is reduced both by extracellular acidosis (as occurs in myocardial ischaemia) and when the drug is applied directly from the intracellular side of the membrane (Du et al., 2011). This study was conducted to gain greater insight into the interaction between flecainide and hERG, by using charged and uncharged flecainide analogues (Liu et al., 2003). Neutral (NU-Flec; pKa 6.3) and fully charged (QX-Flec) flecainide analogues (ASCENTTM Scientific) were tested in whole cell patch-clamp experiments performed at 37oC on HEK293 cells stably expressing hERG. hERG current (IhERG) tails were measured at -40mV after a 2 sec depolarizing step from -80mV to +20mV (N≥5 at each concentration). Normal flecainide produced concentration-dependent inhibition of IhERG with an IC50 value of 1.69µM (CI:1.44-1.99) and a corresponding Hill coefficient of 0.82 (CI:0.70-0.94). When applied extracellularly both analogues showed a slowed, less extensive blocking action compared with flecainide: 6 minutes of continuous superfusion with 100µM NU-Flec inhibited IhERG by only 49.9±4.3% (n=10); while 100µM QX-Flec produced an IhERG inhibition of 45.5±3.5% (n=10). Differences between the two analogues were observed when they were applied to the cell interior via the patch pipette. 100µM of pipette- applied NU-Flec produced little effect on IhERG density (p>0.5, t-test, unpaired comparison); while 10µM or 100µM of internally applied QX-Flec produced a fast and extensive current density reduction of ~80% and ~97% respectively. One interpretation of these findings is that both charged and uncharged flecainide can cross the membrane, but that the charged form seems to be mainly involved in the physical interaction and block of hERG channels from the cytosolic side of the cell membrane.