Voltage-gated K+ channels exhibit a slow inactivation process, which becomes an important influence on the rate of action potential repolarization during prolonged or repetitive depolarization. During slow inactivation, the outer mouth of the permeation pathway undergoes a conformational change. We report here that during the slow inactivation process, the channel progresses through at least three permeation states; from the initial open state that is highly selective for K+, the channel enters a state that is less permeable to K+ and more permeable to Na+, and then proceeds to a state that is non-conducting. Similar results were obtained in three different voltage-gated K+ channels: Kv2.1, a channel derived from Shaker (Shaker Delta A463C), and a chimeric channel derived from Kv2.1 and Kv1.3 that displays classical C-type inactivation. The change in selectivity displayed both voltage- and time-dependent properties of slow inactivation and was observed with K+ on either side of the channel. Elevation of internal [K+] inhibited Na+ conduction through the inactivating channel in a concentration-dependent manner. These results indicate that the change in selectivity filter function is an integral part of the slow inactivation mechanism, and argue against the hypothesis that the inactivation gate is independent from the selectivity filter. Thus, these data suggest that the selectivity filter is itself the inactivation gate.