Replication of plasmid ColE1 is regulated by a plasmid-specified small RNA (RNA I). RNA I binds to the precursor (RNA II) of the primer for DNA synthesis and inhibits primer formation. The process of binding of RNA I to RNA II that results in formation of a stably bound complex consists of a series of reactions forming complexes differing in the stability. Formation of a very unstable early intermediate that was previously inferred from the inhibition of stable binding caused by a second RNA I species was firmly established by more extensive studies. This complex is converted to a more stable yet reversible complex that was identified by its RNase sensitivity, which was altered from that of the earlier complex or from that of free RNA I or RNA II. In these complexes, most loops of RNA II interact with their complementary loops of RNA I. The kinetic and structural analyses of the binding process predict formation of a complex interacting at a single pair of complementary loops that precedes formation of these complexes. Thus the process of binding of RNA I to RNA II is seen to consist of a sequence of reactions producing a series of progressively more stable intermediates leading to the final product.