With prolonged stimulation, voltage-activated K+ channels close by a gating process called inactivation. This inactivation gating can occur by two distinct molecular mechanisms: N-type, in which a tethered particle blocks the intracellular mouth of the pore, and C-type, which involves a closure of the external mouth. The functional motion involved in C-type inactivation was studied by introducing cysteine residues at the outer mouth of Shaker K+ channels through mutagenesis, and by measuring state-dependent changes in accessibility to chemical modification. Modification of three adjacent residues in the outer mouth was 130-10,000-fold faster in the C-type inactivated state than in the closed state. At one position, state-dependent bridging or crosslinking between subunits was also possible. These results give a consistent picture in which C-type inactivation promotes a local rearrangement and constriction of the channel at the outer mouth.