The blocking action of 4-aminopyridine (4-AP) on the cloned K+ channel Kv1.5 expressed in Xenopus oocytes was studied using the two-microelectrode voltage-clamp method. Application of 4-AP to the bath solution reversibly suppressed the expressed current in a voltage- and concentration-dependent manner decreasing with membrane depolarization and with a half-maximal inhibitory concentration of 0.14 mM (at +40 mV). Both block and unblock occurred mainly during a depolarization when channels were activated. With successive depolarizations, 4-AP decreased not only the peak amplitudes of the current in successive pulses, but also the current during a depolarization. Upon washout of 4-AP, the current recovered with successive depolarizations, whereas no recovery of the current was noted in the absence of depolarizations. The extent of block markedly increased with alkalization of the external solution and decreased with acidification. External application of 4-amino-pyridine methiodide, a charged form of a quaternary 4-AP derivative, did not affect the current, but internal application markedly suppressed the current, indicating the drug gained access to the channel from the cytoplasmic side. These data suggest that 4-AP crosses the membrane in its uncharged form and acts from inside of the cell in its charged form, resulting in block of the channels with higher affinity to the open state.