Nuclear extracts were prepared from cells infected with herpes simplex virus type 1 (HSV-1) and fractionated by sucrose gradient centrifugation to identify deoxyribonucleoprotein complexes involved in viral replication. Large amounts of an HSV-1 induced protein with a molecular weight of about 133,000 sedimented as a broad peak in the 25 S region of the gradient and cosedimented with 13 S DNA fragments. The sedimentation of both the protein and DNA decreased upon treatment of nuclear extracts with DNase. This result indicated that the protein and DNA were associated in deoxyribonucleoprotein complexes. The protein was identified as the HSV-encoded major DNA-binding protein ICP8 based on its molecular weight, its association with DNA in nuclear extracts, and its immunoprecipitation with monospecific antiserum and monoclonal antibody to ICP8. Deoxyribonucleoprotein complexes containing ICP8 could be immunoprecipitated from nuclear extracts. When DNA was extracted from these immunoprecipitates, fractionated by agarose gel electrophoresis, transferred to nitrocellulose paper, and hybridized to 32P-labeled HSV-1 or cell DNA, both HSV-1 and cell DNA sequences were identified. Cesium chloride gradient analysis of the immunoprecipitated DNA indicated that duplex DNA was present in the complexes. Thus, the major DNA-binding protein of HSV-1 is associated with both duplex HSV-1 and cell DNA in vivo.