Replication of the Escherichia coli plasmid ColE1 initiates from an RNA primer. This primer is formed by a ColE1 RNA II molecule that remains hybridized to its DNA template in the origin region after transcription. Continued hybridization is inhibited by prior binding to RNA II of another ColE1 transcript, RNA I; and this interaction is regulated by the plasmid-encoded Rom protein. To understand the quantitative aspects of regulation of ColE1 synthesis, we have measured the levels of RNA I, RNA II, and Rom protein in vivo, as well as the half-lives of the RNAs. The intracellular concentrations of RNA I, RNA II, and Rom protein were found to be about 1 μM, 7 nM, and 1 μM, respectively; and the RNAs had half-lives of about 2 min. A simple model derived from these results indicates that the plasmid copy number is little affected by the rate of RNA II synthesis but is strongly dependent on that of RNA I.