The replication frequency of plasmid R1 is determined by the availability of the RepA protein, which acts at the origin of replication to promote initiation. Synthesis of RepA is negatively regulated both at the transcriptional and post-transcriptional levels. Post-transcriptional control is exerted through the action of an antisense RNA, CopA RNA. The target of CopA RNA, CopT RNA, is located in the leader region of the RepA mRNA. Binding between CopA and CopT inhibits repA expression. We have previously presented an in vitro analysis of the binding reaction between CopA and CopT RNAs. In this communication, we extend the in vitro analysis by determining the regions of CopA required for binding, and also demonstrate that binding occurs in at least two steps. The first step is the formation of an initial, transient complex; stem-loop II is the structure in CopA necessary and sufficient for this step. The subsequent step(s), resulting in the formation of a complete duplex, requires a stretch of single-stranded nucleotides located 5' to stem-loop II in CopA, and its counterpart in CopT. We show that the single-stranded region can be positioned on either side of stem-loop II provided that there is a complementary stretch of nucleotides in CopT, indicating that the second step(s) is not sequence-specific. Furthermore, the effects of salt concentration and temperature on the binding reaction indicate that duplex formation occurs through a mechanism of gradual intra-strand breaking and inter-strand formation of hydrogen bonds.