Physical analysis of RNA I, the small antisense RNA which regulates the replication of IncB miniplasmid pMU720, showed that it is a highly structured molecule containing an imperfectly paired stem closed by a 6-base hairpin loop. Mutational studies revealed that a 3-base sequence in the hairpin loop is critical to the interaction between RNA I and its complementary target in the RepA mRNA (RNA II). Furthermore, a 2-base interior loop in the upper stem was found to play an important role in facilitating effective binding between RNA I and RNA II. From these analyses, a model describing the molecular mechanism of binding between RNA I and RNA II is proposed.