ICP8 is the major single-stranded DNA (ssDNA) binding protein of the herpes simplex virus type 1 and is required for the onset and maintenance of viral genomic replication. To identify regions responsible for the cooperative binding to ssDNA, several mutants of ICP8 have been characterized. Total reflection X-ray fluorescence experiments on the constructs confirmed the presence of one zinc atom per molecule. Comparative analysis of the mutants by electrophoretic mobility shift assays was done with oligonucleotides for which the number of bases is approximately that occluded by one protein molecule. The analysis indicated that neither removal of the 60-amino-acid C-terminal region nor Cys254Ser and Cys455Ser mutations qualitatively affect the intrinsic DNA binding ability of ICP8. The C-terminal deletion mutants, however, exhibit a total loss of cooperativity on longer ssDNA stretches. This behavior is only slightly modulated by the two-cysteine substitution. Circular dichroism experiments suggest a role for this C-terminal tail in protein stabilization as well as in intermolecular interactions. The results show that the cooperative nature of the ssDNA binding of ICP8 is localized in the 60-residue C-terminal region. Since the anchoring of a C- or N-terminal arm of one protein onto the adjacent one on the DNA strand has been reported for other ssDNA binding proteins, this appears to be the general structural mechanism responsible for the cooperative ssDNA binding by this class of protein.