1. The mechanism of quinidine‐induced ionic and gating current inhibition was studied in human Kv1.5 (hKv1.5) delayed rectifier channels expressed in human embryonic kidney cells. In the steady state, quinidine produced a voltage‐dependent block between +30 and +120 mV (Kd at +60 mV = 7.2 microM) with an equivalent electrical distance, delta, of 0.29 +/‐ 0.06 and 0.26 +/‐ 0.05 at 10 and 50 microM quinidine, respectively. The apparent affinity at 0 mV (Kd) was 25 microM at 10 microM quinidine and 38 microM at 50 microM quinidine. The data suggested a quinidine binding site that sensed 20‐30% of the transmembrane electrical field, from the inside. Non‐steady‐state measurements indicated rapid open channel block with mean time constants of 2.1 +/‐ 0.9 and 1.2 +/‐ 0.2 ms at 10 and 50 microM quinidine, respectively. 2. ‘On’ gating current (on‐Ig) was unaffected over a wide range of potentials and between 10 and 100 microM quinidine. On‐gating charge (Qon) was similarly conserved in the steady state between ‐100 and +50 mV. On return to ‐100 mV, quinidine slowed the off‐gating current (off‐Ig) after depolarizations more positive than ‐25 mV. After depolarizations to +50 mV, only 59 +/‐ 3.4% (10 microM quinidine) and 6.6 +/‐ 9.5% (100 microM quinidine) of the charge returned within 25 ms, compared with 100% in control. Due to the conservation of Qon in subsequent pulses, the remaining charge must have returned during the subsequent 10 s interpulse interval. 3. A threshold for quinidine action on off‐Ig was established positive to ‐25 mV. The voltage dependence of Qoff immobilization at more positive potentials than +20 mV had an equivalent electrical distance of 0.32 +/‐ 0.04 (10 microM quinidine) and 0.20 +/‐ 0.32 (100 microM quinidine) with calculated Kd values of 21.6 +/‐ 4.6 and 16.2 +/‐ 8.4 microM at 10 and 100 microM quinidine, respectively. These characteristics of block are in good agreement with values obtained from ionic data. 4. Simultaneous measurements of ionic and gating currents confirmed, after subtraction, an ionic current threshold at ‐21.8 +/‐ 1.8 mV. The gating current data confirm directly that ionic current block by quinidine occurs by binding at a site on the hKv1.5 channel that becomes accessible when the channel opens. There was no evidence for action of quinidine on kinetic states prior to the open state at concentrations of quinidine up to 100 microM.