The pore-forming colicins N and A require the porin, OmpF, in order to translocate across the outer membrane of Escherichia coli. We investigated the hypothesis that in vivo, colicins N and A may traverse the outer membrane through the OmpF channel. In order to accommodate a polypeptide in the pore, the mid-channel constriction loop of OmpF, L3, would need to undergo a conformational change. We used five OmpF cystine mutants, which fix L3 in the conformation determined by X-ray crystallography, to investigate L3 movement during colicin activity in vivo. Sensitivity to colicins N and A of E. coli cells expressing these OmpF cystine mutants was determined using cell survival and in vivo potassium efflux and fluorescence assays. Results indicate that gross movement of L3 is not required for colicin N or A activity and that neither of these colicins crosses the outer membrane of E. coli through the lumen of the OmpF pore.