We isolated and characterized mutants of ColE2 with increased copy number (cop) and those with reduced sensitivity to the wild-type incA gene (inc). Both types of mutations were single-base substitutions in the incA region and simultaneously increased the plasmid copy number and reduced the inhibitory activity of the incA gene on Co1E2 DNA replication. Most of the cop mutations also reduced sensitivity to the wild-type incA gene. These mutations were located in the region specifying the large stem-and-loop structures of RNA I and the 5′ portion of the Rep mRNA. All these results indicate that RNA I interacts with the Rep mRNA and thereby inhibits expression of the Rep protein at a post-transcriptional step and that this is probably the only mechanism that controls the CoIl2 Rep protein expression. It is suggested that only portions of the nucleotides in the loop region are involved in initial (kissing) interaction of these RNAs. The total level of rep gene expression in the host cells appears to be kept constant (at a level characteristic for each cop allele) irrespective of the actual plasmid copy number above a certain level, when rep gene expression is regulated by the incA gene on the same plasmid. These seem to be the basic mechanisms for the replication control of Co1E2.