HERG (human ether-à-go-go-related gene) encodes the α-subunit of channels carrying the cardiac rapid delayed rectifier K⁺ current (I_Kr), which is a major determinant of the duration of ventricular action potentials and of the QT interval. This study investigated the effects on HERG channel current (I_HERG) of clemastine, a ‘conventional’ antihistamine that has been associated with delayed ventricular repolarization in vitro [1,2], but for which no adverse effects on human QT interval have been reported. Whole-cell patch-clamp measurements of I_HERG were made at 37°C from human embryonic kidney (HEK 293) cells stably expressing HERG channels [3]; the extracellular solution was normal Tyrode solution, and the pipette solution was a physiological K-based solution. I_HERG tails at -40 mV following two-second depolarizing pulses to +20 mV were inhibited by clemastine with an IC₅₀ value of 12 nM; this drug concentration also inhibited peak I_HERG by 41.1 ± 1.2% (mean ± SEM) when elicited during a voltage-clamp command of a digitised rabbit ventricular action potential. Clemastine produced a reversible ~ -5 mV shift in the I_HERG steady-state voltage-dependent activation curve, but voltage dependence of inactivation was unaffected. Development of I_HERG inhibition by clemastine showed strong time-dependence. The S6 point mutation Y652A greatly attenuated the inhibitory effect of 120 nM (10X IC₅₀) clemastine from 89.4 ± 2.5% block (WT) to 19.3 ± 4.3% (Y652A-HERG ), as did the mutation F656A, which reduced inhibition from 84.5 ± 1.4% block (WT) to 9.0 ± 6.1% block (F656A-HERG), all when using protocols and conditions previously described for these mutant channels [3] (n = 5 or 6 for all, P < 0.001). We conclude that clemastine is a high potency inhibitor of the HERG K⁺ channel, exhibiting characteristics of a preferential open/activated channel blocker and interacting with a high affinity drug-binding site in the HERG channel pore cavity. The disparity between clemastine’s potent I_HERG inhibition and a lack of QT-prolongation in normal clinical use underscores the need to interpret HERG IC₅₀ data for novel compounds in the context of information from other safety assays.