Transposon mutagenesis was used to isolate an Escherichia coli mutant that released into the external medium a heterologous protein, the Aeromonas hydrophila aerolysin. Genetic mapping and phenotypic characterization of E. coli CM209 showed that Tn5 is inserted in the tolQ gene. This mutant strain released a significant amount of unprocessed (proaerolysin) protein into the external medium, together with other periplasmic proteins. Similar levels of the toxin were detected in the intracellular compartments of the parental and mutant strains. Whereas inactivation of the tolQ gene itself was responsible for some of the phenotypic properties of strain CM209, such as tolerance to group A colicins or to filamentous bacteriophages, the leaky phenotype was associated with a polar effect exerted by Tn5 on distal genes of the tolQRA cluster.