Block of hERG1 K+ channels by many drugs delays cardiac repolarization, prolongs QT interval and is associated with an increased risk of cardiac arrhythmia. A common finding is that high affinity binding of potent hERG channel blockers is inactivation state dependent. The most compelling evidence for this assertion is that point mutations in hERG that inhibit or eliminate inactivation gating (e.g., S620T or G628C/S631C) can reduce drug affinity by >100-fold. Here we examine the link between hERG1 inactivation and blocker affinity in more detail by characterization of concatenated tetramers containing a variable number of subunits harboring mutations that disrupt inactivation. For concatenated tetramers containing only wild-type hERG subunits (wt4) the IC50's were 0.20 µM, 0.16 µM, 0.18 µM for cisapride, MK499 and dofetilide, respectively. The presence of a single S620T subunit in a tetramer (ST1/wt3) disrupted inactivation gating (+76 mV shift in V0.5) similar to a homomeric S620T tetramer (ST4; +87 mV shift in V0.5). For ST4 channels, IC50's were 10.6 µM, 6.97 µM, 14.1 µM for cisapride, MK499 and dofetilide, respectively. In contrast, for ST1/wt3 channels the IC50's were 0.60 µM, 1.51 µM, 3.16 µM for the same three drugs. Thus, despite similar effects on inactivation, drug sensitivity of ST4 channels was significantly less than ST1/wt3 channels, indicating that the S620T mutation alters drug binding by a mechanism other than attenuation of inactivation. Unlike S620T, the double mutation G628C/S631C completely eliminates inactivation and concatenated tetramers containing either one or four G628C/S631C subunits induced the same 2.5-fold decrease of cisapride sensitivity (IC50: 0.50 µM and 0.47 µM). Together these findings indicate that preferential binding of drugs to the inactivated state of hERG channels is far less important than is commonly claimed.