RepA, the replication initiator protein of the IncB plasmid pMU720, acts preferentially in cis. The cis activity of RepA is thought to be mediated by CIS, a 166-bp region of DNA separating the coding region of repA from the origin of replication (ori) of pMU720. To investigate the trans activity of RepA, the repA gene, without its cognate ori, was cloned on a multicopy plasmid, pSU39. The ori on which RepA acts was cloned on pAM34, a plasmid whose replicon is inactive without induction by isopropyl--D-thiogalactopyranoside (IPTG). Thus, in the absence of IPTG, replication of the pAM34 derivatives was dependent on activation of the cloned ori by RepA produced in trans from the pSU39 derivatives. The effect of CIS, when present either on the RepA-producing or the ori plasmid or both, on the efficiency of replication of the ori plasmid in vivo, was determined. The presence of CIS, in its native position and orientation, on the RepA-producing plasmid reduced the efficiency of replication of the ori plasmid. This inhibitory activity of CIS was sequence specific and involved interaction with the C-terminal 20 to 37 amino acids of RepA. By contrast, CIS had no effect when present on the ori plasmid. Initiation of replication from the ori in trans was independent of transcription into CIS.