Mutants of Escherichia coli K-12 have been isolated that suppress cer mutants, ColE1 mutants that are unable to replicate as the plasmid. These host suppressors were designated her, for host factor affecting ColE1 replication. Each her suppressor showed a characteristic pattern of suppression depending on the cer mutation used for selecting the mutant bacteria. One of the suppressors, named herA, that suppressed cer6, a single-base-pair alteration 160 base pairs upstream of the ColE1 replication origin, was genetically identified as an alteration of the rnh gene (RNase H). HerA was recessive to its wild-type allele. RNase H activity of herA cell extracts was defective. Conversely, rnh mutants that were isolated independently of ColE1 replication supported replication of cer6 DNA. Some rnh mutants manifested the HerA phenotype only above a certain transition temperature, and their RNase H activity was found to be temperature sensitive. Therefore, replication of cer6 DNA in vivo is sensitive to RNase H activity. Under the conditions that suppressed cer6, the wild-type colE1 replicon replicated normally. Then, ColE1 replication in vivo proceeds in the absence of RNase H activity, which has been shown to be required for in vitro replication of the DNA.