The major DNA-binding protein or infected cell protein 8 (ICP8) encoded by herpes simplex virus exhibits multiple interactions with the cell nucleus in that it interacts with the host cell nuclear matrix and viral DNA molecules as sequential stages in its maturational process (M. P. Quinlan, L. B. Chen, and D. M. Knipe (1984), Cell 36, 857–868). To define the portion(s) of ICP8 required for DNA binding, we have fine-mapped and identified the sequence changes in mutant genes causing changes in the protein that affect DNA binding. These mutations lead to amino acid changes between residues 348 and 450 of ICP8. Construction of a mutant ICP8 gene specifically altered at residues 499 and 502 led to a gene product that was also defective in a nuclear function. Thus, at least part of the region of ICP8 from residues 348 to 4.50 is required for DNA binding by ICP8. This portion of the protein may be involved in binding to DNA or forming intermolecular contacts needed for cooperative DNA binding. If this region is directly involved in binding of the protein to DNA, the most likely structure predicted for this region involves folding of β-strands to form a channel for binding to a nucleotide chain.