We have isolated spontaneous rifampicin-resistant mutants fromEscherichia colithat showed allele-specific suppression of the copy-number phenotype of ColE1 high-copy-number mutantsin vivo.The key step in the regulatory circuitry of the initiation of ColE1 DNA replication is the formation of the persistent hybrid between the primer RNA and the DNA template around the replication origin. Three host-encoded enzymes, RNase H, DNA polymerase I, and RNA polymerase, are essential to the replication initiationin vitro.To decide whether the activity of RNA polymerase is involved directly in the formation of the persistent hybrid, we screened rifampicin-resistant colonies for suppressors of ColE1 copy-number mutants. Suppressor strain YY572 (rpoB572) changes the 572 residue of the β subunit of RNA polymerase, encoded by therpoBgene, from isoleucine to leucine. Another suppressor, YY513 (rpoB513), changes the 513 residue from glutamine to lysine. The other known rifampicin-resistant alleles located at residue 513,rpoB8andrpoB101,did not show a significant suppression of the copy number of those ColE1 copy-number mutants asrpoB513.The suppression byrpoB513on different ColE1 copy-number mutants showed allelic specificity. The possible roles of RNA polymerase in control of ColE1 copy number are discussed.