We have previously found that nicotine blocked multiple K+ currents, including the rapid component of delayed rectifier K+ currents (IKr), by interacting directly with the channels. To shed some light on the mechanisms of interaction between nicotine and channels, we performed detailed analysis on the human ether-à-go-go-related gene (HERG) channels, which are believed to be equivalent to the native I(Kr) when expressed in Xenopus oocytes. Nicotine suppressed the HERG channels in a concentration-dependent manner with greater potency with voltage protocols, which favor channel inactivation. Nicotine caused dramatic shifts of the voltage-dependent inactivation curve to more negative potentials and accelerated the inactivation process. Conversely, maneuvers that weakened the channel inactivation gating considerably relieved the blockade. Elevating the extracellular K+ concentration from 5 to 20 mM increased the nicotine concentration (by approximately 100-fold) needed to achieve the same degree of inhibition. Moreover, nicotine lost its ability to block the HERG channels when a single mutation was introduced to a residue located after transmembrane domain 6 (S631A) to remove the rapid channel inactivation. Our data suggest that the inactivation gating determines nicotine blockade of the HERG channels.