Porins, such as Escherichia coli OmpF, provide the only reported example of a voltage-gated channel where the three-dimensional structure is known to high resolution. Mutations that affect voltage-gating are clustered around the eyelet region, which is a mid-channel constriction caused by a polypeptide loop (L3) folding inside the lumen of this beta-barrel pore. These data, combined with molecular dynamics simulations, indicate that voltage-gating may involve L3 displacement. We have constructed six double cysteine OmpF mutants, five of which form disulphide bonds fixing L3 in the conformation determined by X-ray crystallography. These channels have altered single-channel conductances but unimpaired voltage-gating. The data show that L3 movement is not required for voltage-gating.