The hERG protein is the pore-forming α-subunit of channels responsible for the cardiac IKr potassium current and is sensitive to pharmacological inhibition by Class I and Class III antiarrhythmic agents. Previous studies have demonstrated differences between these two classes of compounds regarding the dependence of hERG current (IhERG) inhibition on the channel’s uniquely fast inactivation process. In particular, Class Ia (e.g. disopyramide and quinidine) and Ic (propafenone) agents were found to be less dependent on inactivation for channel block to occur, while Class III methanesulphonanilide compounds (e.g. dofetilide and E-4031) were found to be more dependent on hERG channels inactivation (Ficker et al., 1998; Perrin et al., 2008; McPate et al., 2008). Flecainide is a Class Ic agent widely used for the clinical treatment of atrial fibrillation; it inhibits IhERG at clinically relevant concentrations (Paul et al., 2002). Although flecainide blockade of hERG current (IhERG) has been investigated previously, its sensitivity to channel inactivation has not yet been systematically investigated. Thus, the aim of this study was to evaluate the extent to which flecainide inhibition of IhERG depends on hERG channel inactivation. The drug was tested on different “attenuated-inactivation” mutants: V625A, S631A, N588K and the S631A/N588K double mutant (McPate et al., 2008; Siebrands and Friederich, 2007). IhERG was recorded from hERG-expressing HEK293 cells using whole-cell patch-clamp at 37oC. IhERG tails were measured at -40mV or at -120mV after a 2sec depolarizing step from -80mV to +20mV (n≥5 at each concentration). Flecainide produced a concentration-dependent inhibition of wild-type (WT) IhERG with an IC50 of 1.49µM (CI:1.27-1.74). The IC50 for inhibition of S631A-IhERG [7.49µM (CI:6.33-8.87)] was ~5-fold that for WT-IhERG, whilst that for N588K-IhERG [6.50µM (CI:5.37-7.88)] was ~4.4-fold that of WT-IhERG. The S631A/N588K double mutant leads to a greater attenuation of IhERG inactivation than either individual mutation (McPate et al., 2008) and it exhibited an IC50 [19.16µM (CI:15.43-23.80)] ~12.9 fold that of WT-IhERG. Similarly to S631A/N588K, V625A is characterized by a strong reduction of channel inactivation (Siebrands and Friederich, 2007) and IhERG carried by this mutant was inhibited with an IC50 [28.88µM (CI:23.41-35.61)] ~27.5 fold that of its WT-IhERG control. When compared with prior results (Mc Pate et al., 2002, Perrin et al., 2008) these findings are suggestive that flecainide blockade of IhERG depends on inactivation to a slightly greater extent than Class Ia agents and propafenone, but to a much lesser extent than that of high affinity methanesulphonanilides.