The replication frequency of plasmid R1 is controlled by an antisense RNA, CopA, that inhibits the synthesis of the replication initiator protein, RepA, at the post-transcriptional level. This inhibition is indirect and affects translation of a leader peptide reading frame (tap). Translation of tap is required for repA translation (Blomberg et al., 1992). Here we asked whether an RNA stem-loop sequestering the repA ribosome-binding site blocks tap translation-independent repA expression. Destabilization of this structure resulted in tap-independent RepA synthesis, concomitant with a loss of CopA-mediated inhibition; thus, CopA acts at the level of tap translation. Structure probing of RepA mRNAs confirmed that the introduced mutations induced a local destabilization in the repA ribosome-binding site stem-loop. An increased spacing between the repA Shine-Dalgarno region and the start codon permitted even higher repA expression. In Incl alpha/IncB plasmids, an RNA pseudoknot acts as an activator for rep translation. We suggest that the regulatory pathway in plasmid R1 does not involve an activator RNA pseudoknot.