Two negatively acting functions - the CopA-RNA and the CopB protein - are involved in the control of replication of plasmid R1. They both act as inhibitors of expression of a gene, repA, which seems to be positively required for autonomous plasmid replication. Here we show that the two control functions act separately and independently. The CopB protein represses initiation of transcription of the repA gene, and its target site lies within a 60 base pair region containing the repA promoter. The CopA-RNA acts downstream of the repA promoter in the leader sequence containing the copA gene itself, preceding the repA structural gene. Measurements of RepA-beta-galactosidase expression from wild-type and a copA mutant fusion hybrid in the presence of extra copies of the respective copA genes show that a point mutation affecting the activity of the CopA-RNA can also affect CopA target properties. It is therefore concluded that the target site for the CopA-RNA resides within the copA gene in a small region encoding the loop of a stem-loop structure in the CopA-RNA. In addition, the data indicate a direct nucleic acid-nucleic acid interaction as the basis for the CopA inhibitor activity.