A transcript (RNA II) from the ColE1 primer promoter forms a persistent hybrid with the template DNA near the replication origin. The hybridized RNA II is cleaved by RNAase H to form the primer. Binding of RNA I to RNA II inhibits the hybridization and thus prevents primer formation. The binding is demonstrated by identification of the product. Analyses of the product with RNAase T1 revealed that pairing initiates at or near the 5' end of RNA I and propagates toward its 3' end. Removal of five nucleotides from the 5' end of RNA I greatly reduces its binding activity. Prior to initiation of pairing, RNA I interacts with RNA II at their single-stranded loops. This interaction is transient but facilitates complete pairing along the entire length of RNA I that initiates from the 5'-end region.