human Ether-à-go-go Related Gene (hERG) channels mediate cardiac IKr potassium current which plays a key role in cardiac repolarization and is susceptible to pharmacological inhibition by a range of different compounds, including Class I antiarrhythmic agents. Flecainide is a Class Ic antiarrhythmic agent widely used in the clinical treatment of atrial fibrillation; it blocks IhERG/IKr at clinically relevant concentrations (Breindahl 2000; Paul et al. 2002) and, rarely, can induce life-threatening ventricular tachycardia (Torsade de Pointes), associated with rate corrected QT interval prolongation that is not fully accounted for by widening of the QRS complex (Thevenin et al. 2003; Oguayo et al. 2014). Although hERG block by flecainide has previously been reported (Paul et al. 2002), the flecainide binding site on hERG has not yet been identified. The aim of this study was to determine which residue(s) in the hERG channel pore is/are responsible for the interaction. Flecainide was tested on alanine-mutants of hERG transiently expressed in HEK293 cells. hERG current (IhERG) was recorded using whole-cell patch-clamp at 37°C. Concentration response relations from which half maximal inhibitory concentrations (IC50) were derived, were produced from application of multiple drug concentrations (N≥5 at each concentration). The selected residues included V625, G648, Y652 and F656, all known to be determinants for hERG block, located within the pore region of the channel (Mitcheson et al. 2000). IhERG tails were elicited at -40mV or -120mV after a depolarizing step from -80mV to +20mV. GOLD docking software was then employed to simulate the interaction between flecainide and a MthK-based hERG homology model. Docking outputs were ranked according to their energy scores and the best poses (lowest energy scores) were selected for further analysis. Flecainide was ~142-fold less potent on F656A (compared to its WT hERG in the same experimental condition) with an IC50 of 176.9µM (95% CI: 143.9-217.3) with an nH of 0.55 (95% CI: 0.47-0.63); whilst the mutation of the second aromatic residue Y652A had only a small effect on flecainide block with an IC50 of 5.09µM (95% CI: 4.00-6.46) with an nH of 0.60 (95% CI: 0.53-0.68), corresponding to just ~3.4-fold decrease in potency (versus its WT control). G648A and V625A mutants had a smaller effect than F656A on flecainide potency, shifting the IC50 by ~9 and ~28-fold respectively versus WT under similar conditions. Docking simulations supported these results: in the best low energy score poses, flecainide docked low into the inner mouth of the pore cavity, with the benzamide and piperidine moieties of flecainide making multiple contacts (π-π and cation-π stacking) mainly with the aromatic ring of F656.