The hERG potassium channel is well established to control ventricular repolarisation and to be the pharmacological (anti-)target of many drugs that cause acquired long QT syndrome.  The aromatic S6 domain residues (F656 and Y652) are known to be important blocking determinants for many drugs; an S5 aromatic residue (F557) has recently been proposed to be an additional significant binding determinant (Saxena et al, 2016).  F557 lies close to Y652 (Saxena et al, 2016; Helliwell et al, 2018).  The aim of this study was to exploit drugs with unusual characteristics of hERG inhibition to further probe the role of the F557 residue. Whole-cell patch-clamp recordings of hERG current (IhERG) were made at 37°C from Human Embryonic Kidney (HEK 293) cells either stably or transiently transfected with wild-type (WT) or mutant hERG channels.  A voltage step protocol, comprised of a 2-second depolarization to + 20 mV, was followed by repolarization to −40 mV to elicit IhERG tails, which were measured to quantify drug block (Helliwell et al, 2018). BeKm-1 scorpion toxin exhibits preferential closed channel block (Milnes et al, 2003), consistent with extracellular binding.  At 100nM, BeKm-1 inhibited WT and F557L IhERG tails by 57±7 % (mean ± SEM; n=7 cells) and 55±5 % (n=7 cells; p >0.05 vs WT, unpaired t test ).  Two other concentrations (10 nM and 300 nM) of BeKm-1 also produced levels of IhERG inhibition that did not differ between WT and F557L channels. The macrolide antibiotic erythromycin is large and appears only to have limited access to the S6 aromatic binding site (Duncan et al, 2006). At 100µM, erythromycin inhibited WT IhERG tails by 75±5 % (n=10) and those carried by F557L by 75±4% (n=6; p>0.05 vs WT, unpaired t-test).  A lack of significant difference of inhibitory actions between WT and F557 hERG was also observed for 1 mM erythromycin. IhERG block by the antiarrhythmic propafenone depends on the F656 but not Y652 residue (Witchel et al, 2004). At 300nM propafenone inhibited WT IhERG tails by 45±4%(n=11) and those carried by F557L by 46±7% (n=9, p>0.05 vs WT, unpaired t-test). A lack of significant difference of inhibitory actions between WT and F557 hERG was also observed for 100nM, 1µM and 3µM of propafenone.  As anticipated, a canonical inhibitor, cisapride was significantly less potent against F557L than against WT IhERG (exhibiting a 9-fold greater half-maximal inhibitory concentration for F557L).  Collectively, our findings complement and extend prior studies of the F557 residue, as they argue against a dependence of pharmacological block on this site independent of a compound’s reliance on interaction with Y652 (as occurs with canonical inhibitors such as cisapride (Saxena et al, 2016)).