Voltage-gated channels undergo a conformational change in response to changes in transmembrane voltage. Here we use site-directed biotinylation to create conformation-sensitive sites on colicin Ia, a bacteriocidal protein that forms a voltage-sensitive membrane channel, which can be monitored by electrophysiological methods. We investigated a model of gating developed for the partly homologous colicin E1 that is based on the insertion of regions of the protein into the membrane in response to cis-positive voltages. Site-directed cysteine mutagenesis, followed by chemical modification, was used to attach a biotin molecule covalently to a series of unique sites on colicin Ia. The modified protein was incorporated into planar lipid membranes, where the introduced biotin moiety served as a site to bind the water-soluble protein streptavidin, added to one side of the membrane or the other. Our results show that colicin gating is associated with the translocation across the membrane of a segment of the protein of at least 31 amino acids.