The plasmid pJB01 contains a single operon consisting of three orfs, copA, repB and repC cistrons. The operon, also called repABC operon, starts transcription at T695 or A696 on the pJB01 genetic map. CopA (called RepA in pMV158 family) or ctRNA (counter-transcript RNA) of this plasmid play roles as a repressor of RepB, a replication initiator, on the transcriptional and translational level, respectively. RepC did not bind 73 bp PCR product including three tandem repeats (5′ ′ ′ ′-CAACAAA-3′ ′ ′ ′), the binding sites for RepB and any other regions on pJB01. However, when RepB and RepC were added simultaneously in the reaction mixture for gel mobility shift assay, unexpectedly, three kinds of retarded bands were observed. It suggests that RepC can interact with RepB by protein-protein interaction. In addition, the copy numbers of RepC-deleted pJB01 ermC (erythromycin-resistant methylase C) plasmids are increased 1.37-1.45 folds when compared with that of parent pJB01 ermC. From these, it could be proposed that RepC plays a role as a negative regulator to modify RepB function in the initiation of pJB01 replication, and therefore, the copy number of pJB01 is maintained via mutual global regulation of various replication factors, such as CopA, ctRNA, RepB and RepC.