RNase H activity increases markedly after bacteriophage T4 infection of Escherichia coli MIC2003, an RNase H-deficient host. We have extensively purified the RNase H from these T4-infected cells and have shown that the RNase H activity copurifies with a 5' to 3' DNA exonuclease activity. The N-terminal sequence of a 35-kDa protein copurifying with the RNase H activity matches the terminus of the predicted product of an open reading frame (designated ORF A or 33.2) upstream of T4 gene 33, identified previously by Hahn and co-workers (Hahn, S., Kruse, U., and Rüger, W. (1986) Nucleic Acids Res. 14, 9311-9327). Plasmids containing ORF A under the control of the T7 promoter express RNase H and 5' to 3' DNA exonuclease activities as well as a protein that comigrates on sodium dodecyl sulfate-polyacrylamide gels with the 35-kDa protein present in the RNase H purified from T4-infected cells. T4 RNase H removes the pentamer RNA primers from DNA chains initiated by the T4 primase-helicase (gene products 61 and 41). Addition of T4 RNase H and T4 DNA ligase leads to extensive joining of discontinuous lagging strand fragments in the T4 DNA replication system in vitro.