4-Aminopyridine (4AP) can block various K channels with different state dependences; block occurs in the activated state or in the closed state. The use of K channel clones to study the mechanism and structural determinants responsible for the state dependence of 4AP actions has been hampered by the fact that, for all the K channel clones examined so far, 4AP binding and unbinding occur mainly in the activated state. We report here that 4AP binding to a fast inactivating K channel encoded by Kv4.2 in Xenopus oocytes occurred exclusively in the closed state. The binding rate was slow and independent of membrane voltage in the range from -80 to -120 mV. The binding rate was linearly related to 4AP concentration, yielding apparent binding and unbinding rate constants of 0.012 mM-1 s-1 and 0.062 s-1, respectively. 4AP dissociation from Kv4.2 occurred in two processes, a slow process in the closed state (in a voltage range from -70 to -40 mV) and a fast process in the activated state, which suggested that the closure of the activation gate of Kv4.2 did not prevent the entry or exit of 4AP molecules but slowed these processes. 4AP slowed the rate of Kv4.2 decay during depolarization, consistent with the notion that channel inactivation occurred only after 4AP dissociation. Inactivating Kv4.2 channels prevented 4AP binding. Therefore, 4AP binding and Kv4.2 inactivation were mutually exclusive. This, in conjunction with the observation that 4AP blocked Kv4.2 channels from the intracellular side of the cell membrane, suggests that the 4AP binding site is on the cytoplasmic surface of the Kv4.2 channel at, or adjacent to, the domains involved in channel inactivation. The distinct features of 4AP actions on the time course of transient outward current in human ventricular myocytes suggest that Kv4.2-like subunits are important in the formation of these channels in human heart.