The results presented here locate the gene encoding an early, nonvirion, single-stranded DNA-binding protein of human and simian strains of cytomegalovirus (CMV) [HCMV(Towne) DB140 and SCMV(Colburn) DB129, respectively] and provide additional evidence that this protein is the CMV homolog of the herpes simplex virus type 1 (HSV-1) major DNA-binding protein (ICP8), as proposed earlier (D. G. Anders, A. Irmiere, and W. Gibson, J. Virol. 58:253-262). The ICP8 gene was used as a probe in Southern analyses done at moderate stringency as an approach to locating similar sequences in the CMV genome. The BamHI K and EcoRI V fragments from the center of the long unique segment of HCMV(Towne) hybridized with the ICP8 probe and were in turn used to identify corresponding sequences in the EcoRI D fragment of SCMV(Colburn). RNA prepared from SCMV(Colburn)-infected cells directed the in vitro synthesis of DB129. If the RNA was first hybridized with the cloned 12.5-kilobase EcoRI D fragment, in vitro synthesis of DB129 was specifically inhibited. Additional hybrid-arrested in vitro translation experiments with subclones spanning the EcoRI D fragment demonstrated that the DB129 gene is located in the left half of that fragment, approximately bisected by a SalI site. RNA analyses identified 3.9-, 8.9-, and 10.0-kilobase RNA species expressed from this region. A partial nucleotide sequence of the Colburn region mapping within the boundaries of the 3.9-kilobase transcript, suspected to be the primary coding species, showed significant sequence similarity to the major DNA-binding protein gene homolog identified in B95-8 Epstein-Barr virus.