Escherichia coli rnh mutants were isolated using localized mutagenesis and selective measurements of RNase H activity in mutagenized cell extracts with [3H]poly(rC) X poly(dG) as substrate. RNase H activity in extracts of one mutant, ON152 (rnh-91), was undetectable (less than 0.05% of that of wild-type cells). This mutant formed small colonies at 43 degrees C. At this temperature, accumulation of nascent fragments was more prominent in the rnh-91 X polA4113 double mutant than in the polA4113 mutant; however, no accumulation was found in the rnh single mutant at 43 degrees C. Unlike the 1-3 nucleotide primer RNA found on nascent fragments of polA4113 cells, primers from the rnh-91 X polA4113 cells ranged from one to about ten bases. These results suggest that the 5' leads to 3' exonuclease activity of DNA polymerase I plays a major role in removal of primer RNA and that RNase H functions in an auxiliary role, excising the 5'-portion of longer primers. The rnh mutant supports replication of ColE1-type plasmids. A possible mechanism of replication of such plasmids in rnh mutants and a role of RNase H in the initiation of chromosomal replication are discussed.