A non-self-transmissible multiple-copy plasmid, pEC3, isolated from the phytopathogenic bacterium, Erwinia carotovora subsp. carotovora, can be mobilized with the help of a self-transmissible plasmid. When donors carrying a cya or a crp mutation were used, the mobilization efficiencies of pEC3 were markedly decreased. The covalently closed circular conformation of plasmid DNA containing both oriT and mob genes of pEC3 was found to be relaxed after the addition of cyclic AMP (cAMP). The relaxed DNA contained a specific single-strand DNA nick within the oriT region. RNA transcription of the mob operon was also increased by the addition of cAMP. These results indicate that the expression of the mob operon of pEC3 is positively regulated by cAMP and that the mob gene products are involved in nicking within the oriT region.