1. The relationship between ionic current inactivation and immobilization of 'off'-gating charge in human Kv1.5 channels expressed in human embryonic kidney (HEK293) cells was studied using 4-aminopyridine (4-AP) and tetraethylammonium chloride (TEA-Cl). 2. The charge transferred during short (< 10 ms) depolarizations (Q(on)) was conserved on repolarization (Q(off)) although peak off-gating current (off-Ig) was reduced and the time course prolonged (tau decay increased from 0.4 to > 1.2 ms). For +80 mV pulses longer than 50 ms, Q(off) at 20 ms was less than Q(on) (Q(off)/Q(on) ratio was 0.26 +/- 0.06 at 450 ms). We attribute this to a relative 'immobilization' of gating charge during long depolarizations. 3. 4-AP (0.1-1 mM) prevented slowing of off-Ig, allowing saturation of peak off-Ig. 4-AP also completely prevented immobilization of off-Ig after long depolarizations. In 1 mM 4-AP, off-Ig waveforms decayed rapidly and the charge ratio Q(off)/Q(on) remained at 1.0. 4. In addition to its effects on Ig, 1 mM 4-AP prevented the slow inactivation of ionic current seen during strong depolarizations. An initial block was caused by 4-AP or 1 mM intracellular TEA internally applied. However, only 4-AP prevented the slower, later development of C-type inactivation. 5. We suggest that slow current inactivation is accompanied by a gating charge immobilization in Kv1.5. 4-AP potently inhibits the changes in Q(off)/Q(on0, off-Ig, and ionic currents that underlie slow inactivation. Some actions of 4-AP appear independent of its properties as a blocker of open K+ channels, and are not mimicked by internal TEA.