The human ether-a-go-go-related gene (HERG) encodes a K+ channel with biophysical properties nearly identical to the rapid component of the cardiac-delayed rectifier K+ current (I(Kr)). HERG channels are one primary target for the pharmacological management of arrhythmias. In this study, we investigated the acute effects of the class III antiarrhythmic drug amiodarone on HERG channels expressed heterologously in Xenopus oocytes by use of the two-microelectrode voltage clamp technique. Amiodarone blocked HERG channels with an IC50 of 9.8 microM with a maximum outward tail current reduction of 62.8%. The block consisted of two main components, a closed channel block that could not be reversed within the time of experiments and an open channel block with a slow unblock, having a recovery time constant of 73 s at -80 mV. Inactivation of the HERG channel at very positive potentials could not prevent amiodarone block. These results indicate that HERG channels can be blocked by amiodarone in closed, open and inactivated states. The block of open channels was cumulative, use-dependent and voltage-dependent. In summary, our data suggest that the strong class III antiarrhythmic action of amiodarone is at least partially based upon its acute inhibitory effects on HERG potassium channels.