The effect of 4-aminopyridine (4-AP) on membrane and gating currents of Kv1.5 channels was studied in a human cell line. The rank order of block was cell-attached > whole cell >> outside-out macropatches, which suggested that 4-AP blocked channels from the cytosolic face. Stimulation after exposure to 4-AP during rest resulted in a "supernormal" current immediately after the onset of depolarization, followed by open channel block during maintained depolarization. 4-AP remained trapped in closed channels, and unblock after drug washout required depolarization. At higher concentrations, 4-AP induced channel block by binding to closing or nonconducting channels. This effect could be reduced by hyperpolarization and higher pulsing rates. Block of Kv1.5 channels under steady-state conditions was correlated with a dose-dependent reduction in gating charge movement. The normalized voltage dependence of gating was shifted to more negative potentials by 4-AP. A model incorporating both open and closed channel binding of 4-AP reproduced data, including the "supernormal" current, by shifting the Po - V curve slightly to the left. The results show that 4-AP can both enhance and block Kv1.5 current and suggest that 4-AP can bind with different affinities to more than one site on Kv1.5 channels.