The clinical use of the antipsychotic drug thioridazine (THIO) is associated with a significant risk of prolongation of the rate-corrected QT interval of the ECG (Reilly et al. 2000). Most QT-prolonging drugs are associated with pharmacological blockade of the cardiac ‘rapid’ delayed rectifier K⁺ current (I_Kr) and the current (I_HERG) carried by its cloned equivalent, HERG. THIO has been identified as an I_Kr/HERG blocker (Drolet et al. 1999). The aim of this study was to investigate the mechanism of I_HERG blockade by THIO. Whole-cell patch-clamp experiments were performed at 37°C on Human Embryonic Kidney (HEK293) cells expressing either wild-type (WT) HERG or one of two S6-pore HERG-mutants:Y652A or F656A (these residues have been shown to be critical molecular determinants of HERG block by a number drugs). Cells were superfused with a physiological (Na⁺-based) Tyrode’s solution, and the pipette solution was a K⁺-based intracellular salt solution. THIO blocked WT-HERG IHERG with an IC₅₀ of 91 nM (95% CI. 64.9-129.2 nM, 4 concentrations between 10nM and 5μM tested; n=4-5 at each) and Hill slope = 0.74 ± 0.04. Following equilibration in THIO (at either 0.1 or 1μM) block of I_HERG developed rapidly following onset of a sustained depolarization from -80mV to 0mV, consistent with an activation-dependent mechanism. The HERG S6 mutation Y652A partially attenuated HERG block by 1μM THIO, from 87 ± 1% (n=5) in WT-HERG to 55 ± 1% (n=5; t-test p<0.05). The S6 mutation F656A was highly resistant to 1μM THIO (a blockade of only 3 ± 5% was observed; n=5). Collectively, these data are consistent with THIO being a high potency, open-channel HERG blocker binding within the channel pore cavity and with F656 being a critical determinant of drug binding and Y652 also having some influence.