The Class Ia antiarrhythmic drug disopyramide (DISO) causes QT interval prolongation that is potentially dangerous in acquired Long QT Syndrome but beneficial in short QT syndrome, through inhibition of the hERG-encoded channels responsible for rapid delayed rectifier K(+) current (I(Kr)). In this study, alanine mutants of hERG S6 and pore helix residues and MthK-based homology modelling and ligand docking were used to investigate molecular determinants of DISO binding to hERG. Whole-cell hERG current (I(hERG)) recordings were made at 37°C from HEK-293 cells expressing WT or mutant hERG channels. WT outward I(hERG) tails were inhibited with an IC(50) of 7.3μM, whilst inward I(hERG) tails in a high [K(+)](e) of 94mM were blocked with an IC(50) of 25.7μM. The IC(50) for the Y652A mutation was ~55-fold that of WT I(hERG); this mutation also abolished a leftward shift in voltage-dependent I(hERG) activation present for WT hERG. The IC(50) for F656A I(hERG) was ~51 fold its corresponding WT control. In contrast to previously studied methanesulphonanilide hERG inhibitors, neither the G648A S6 nor the T623A and S624A pore helical mutations modified DISO IC(50). Computational docking with the hERG model showed that DISO did not exhibit a single unique binding pose; instead several low energy binding poses at the lower end of the pore cavity favoured interactions with Y652 and F656. In the WT hERG model DISO did not interact directly with residues at the base of the pore helix, consistent with the minimal effect of mutation of these residues on drug block.