After removal of N-type inactivation in Shaker K channels another inactivation process remains (C-type inactivation). The C-type inactivation time course is reversibly slowed when external [K+] increases. The effect of K+ is mimicked by Rb+ and, with less potency, by the less permeant ions Na+, Cs+, and NH4+. These results, which can be explained by the foot-in-the-door model of gating, could reflect the variable interaction of cations with amino acids in the ion-conducting pore. Mutations at position 449 (near the outer mouth of the pore) produce drastic changes in C-type inactivation kinetics and in its interaction with monovalent cations. Replacement of threonine in the wild-type by glutamic acid or lysine leads to a hundred-fold acceleration of inactivation (time constant approximately 25 ms). In contrast, placing valine at this position results in channels that do not inactivate in 45 s. Moreover, high K+, besides slowing down the inactivation kinetics, produces an increase in current amplitude despite a concomitant decrease in K+ driving force. This second effect, which is larger in mutants with faster inactivation kinetics, is caused by an increase in the number of channels that open on depolarization. Thus, C-type inactivation is a process influenced by the ionic composition of the external milieu which strongly depends on the amino acid at position 449 in the pore region. These findings may help to explain the variability in inactivation kinetics observed in the various types of K channels.