An in vitro replication system has been used to study the control of DNA replication of the relaxed plasmids Col E1 and RSF1030. An RNA transcript approximately 100 nucleotides long is synthesized during the in vitro DNA replication reaction. This RNA is synthesized approximately 450 bp away from the origin of replication. A small insertion in the coding sequence for the RNA made from Col E1 DNA leads to a larger RNA species and simultaneously to an increase in plasmid copy number. Revertants missing the specific insertion show shorter RNA transcripts and wild-type copy number. Although plasmids Col E1 and RSF1030 have no extensive sequence homology, the RNA synthesized during RSF1030 replication has almost the same mobility as the Col E1 RNA on polyacrylamide gels and hybridizes to the Col E1 origin region. Extracts prepared from mutants of Escherichia coli deficient in ribonuclease III do not replicate RSF1030 or Col E1 plasmids in vitro. When supplemented with homogeneous RNAase III, such extracts do support DNA replication on these templates, indicating that RNAase III is required for DNA replication. We propose that the 100 nucleotide RNA species is involved in regulating the initiation of DNA replication of these plasmids, and that RNAase III may be involved in processing this RNA.