Protease digestion of the herpes simplex virus type I major single-strand DNA binding protein ICP8 showed that the cleavage patterns observed in the presence and absence of single-stranded DNA oligonucleotides are substantially different with protection of cleavage sites between amino acids 293 and 806 observed in the presence of oligonucleotide. Experiments using ICPS modified with fluorescein-5-maleimide (FM) showed that the fluorescence signal exhibited increased susceptibility to antibody quenching and a significant decrease in polarization of the FM fluorescence was observed in the presence compared to the absence of oligonucleotide. Taken together, these results indicate that ICP8 undergoes a conformational change upon binding to single-stranded DNA.