The movement of some residues in the inner and outer pore region of the hKv1.3 channel causes its characteristic C-type inactivation. To get more details about the spatial arrangement of the inactivated state, we introduced cysteine mutations and assessed the influence on the current by applying extracellular MTSEA, a cysteine modifying reagent. Former studies showed that structural changes of the inner and outer pore during gating had an effect on the accessibility of the cysteine side chains. Amino acids L418C and T419C (Shaker positions 468, 469) in the S6 segment could only be modified by MTSEA in the open but not in the inactivated or closed state whereas the cysteines at positions V417C and I420C (Shaker positions 467, 470) could be modified in the open and inactivated state and not in the closed state. In an attempt to find those structures of the channel that prevented MTS modification in the inactivated L418C mutant channel we examined the L346C (Shaker positions 396) in the S5 segment which is in close proximity to L418. Currents through hKv1.3_L346C channels had similar characteristics as currents through wild-type hKv1.3 channels and could –like wild type channels- not be modified by externally applied MTSEA. The double mutant hKv1.3_L346C_L418C channel could be modified by MTSEA in the open but not in the inactivated and closed state indicating that the L346C mutation does not alter the modification behavior of hKv1.3_L418C. We conclude that the relative movement of the S6 segments that occurring during C-type inactivation that also includes a movement of the side chains of the amino acid at position 418 in hKv1.3 channels was not disturbed by the L346C mutation. This work was supported by a grant from the DFG (Gr848/17-1).