SummaryThe mechanisms ensuring regulation of DNA replication in genomes containing multiple replicons are poorly understood. In this report, we addressed this question by analysing in Bacillus subtilis the replication of a derivative of the promiscuous plasmid pIP501 that carries a rolling-circle and a theta replicon. Genetic analyses revealed that the rolling-circle replicon is strongly inhibited in the derivative and that inhibition requires three elements involved in theta replication: the replication origin, the initiator RepR protein and strong transcription of the repR gene. Inhibition is, however, independent of DNA synthesis at the theta origin. We conclude that rolling-circle inhibition is caused by an inhibitor y signal encoded by the theta replicon and propose that the signal is composed, at least, of the RepR protein bound to its cognate origin.