The synthesis of the rate-limiting RepA replication initiator protein of plasmid R1 is negatively controlled by an antisense RNA, CopA. The regulation is posttranscriptional and involves an inhibitory effect on RepA translation mediated by the binding of CopA to its target (CopT) in the leader region of the RepA mRNA. The evolutionary conservation of the intergenic region between the copA gene and the repA reading frame among plasmids related to R1 may be indicative of an important function in this regulation. One possibility is that sequences/structures in this region might be required for the presumed distal effect of CopAQCopT binding. We have performed a mutational analysis of this region, starting with a mutant repA-lacZ fusion plasmid that shows decreased RepA-LacZ synthesis compared to a wild-type construct, and have identified five compensatory mutations that increase repA-lacZ expression. Two of these were single base-pair substitutions in the copA promoter leading to a decrease in CopA transcription. The other three mutations increased RepA synthesis in the presence as well as in the absence of functional CopA. Reconstructed plasmids carrying these mutations--in conjunction with the original down-mutation or in an otherwise wild-type background--show the expected increase in copy number. The effect of two of these mutations is consistent with the destabilization of a putative secondary structure which may be responsible for the normally low translation rate of the RepA reading frame. The implications of the types of mutations found in this study, as well as the absence of other classes of mutations, are discussed in terms of alternative possible models of CopA-mediated inhibition of RepA synthesis.