Colicin A is a pore-forming bacteriocin that depends upon the Tol proteins in order to be transported from its receptor at the outer membrane surface to its target, the inner membrane. The presequence of yeast mitochondria cytochrome c1 (pc1) as well as the first 167 amino acids of cytochrome b2 (pb2) were fused to the pore-forming domain of colicin A (pfColA). Both hybrid proteins (pc1-pfCoIA and pb2-pfColA) were cytotoxic for Escherichia coli strains devoid of colicin A immunity protein whereas the pore-forming domain without presequence had no lethal effect. The entire precursors and their processed forms were found entirely associated with the bacterial inner membrane and their cytotoxicities were related to their pore-forming activities. The proteins were also shown to kill the tol bacterial strains, which are unable to transport colicins. In addition, we showed that both the cytochrome c1 presequence fused to the dihydrofolate reductase (pc1-DHFR) and the cytochrome c1 presequence moiety of pc1-pfCoIA were translocated across inverted membrane vesicles. Our results indicated that: (i) pc1-pfCoIA produced in the cell cytoplasm was able to assemble in the inner membrane by a mechanism independent of the tol genes; (ii) the inserted pore-forming domain had a channel activity; and (iii) this channel activity was inhibited within the membrane by the immunity protein.