Three Clo DF13 mutant plasmids (designated as clp03, clp05 and clp21) that show a decreased cloacin activity were isolated. The decreased cloacin activity was not due to a reduced number of Clo DF13 copies per cell. The cloacins produced by the clp03 and the clp21 mutant plasmids have a strongly decreased killing activity in vivo in comparison with the wild type cloacin and the cloacin of the clp05 mutant plasmid. Furthermore no lacunae could be observed from clp03 or clp21 harbouring strains, while strains harbouring the clp05 plasmid showed a 50-100 times decreased frequency of lacunae. In addition the clp05 mutant showed a decreased rate of RNA synthesis in clp05 harbouring Escherichia coli minicells. No complementation between the three mutant plasmids was observed. We suggest that the clp03 and clp21 mutations are located in the gene coding for the cloacin. Since the cloacin produced by the clp05 mutant plasmid has retained all the known wild type cloacin activities, the reduced inhibition zone in the stab test is probably caused by a mutation affecting the expression of the cloacin gene. The nature of this mutation is discussed.