The DNA-binding protein, ICP8, of herpes simplex virus type 1 (HSV-1) is multifunctional in vivo and binds preferentially to single-stranded DNA (ssDNA) in vitro. To define the ssDNA-binding domain of ICP8, peptides were produced and analyzed. Portions of the ICP8 gene were cloned into the transcription vector pSP64, and RNA was synthesized in vitro. Translation of this RNA in rabbit reticulocyte lysates produced peptides of 29, 35 and 30 kDa, representing amino-acid residues 332-564, 571-899 and 900-1196, respectively, of intact ICP8 (128 kDa, 1196 amino acids). These peptides were analyzed by ssDNA-cellulose column chromatography. About 55% of the 29 kDa peptide bound to ssDNA-cellulose columns, and the majority which bound eluted with 1.0 M NaCl. About 5% of the 35 kDa peptide and 12% of the 30 kDa peptide bound and eluted with 0.3 M NaCl. Thus, three regions of ICP8 were associated with ssDNA-binding activity. The ssDNA-binding domain of ICP8 was not completely defined, however, because a 95 kDa peptide which included these regions did not bind to or elute from ssDNA-cellulose in the same way as intact ICP8. Amino-acid residues 332-564 and 571-899 not only were associated with ssDNA-binding activity but also contain the altered amino acids of four ICP8 molecules which are deficient in DNA binding.