We had previously demonstrated that the herpes simplex virus 1 (HSV-1) single-stranded DNA-binding protein (ICP8) can specifically stimulate the helicase activity of the HSV-1 origin-binding protein (UL9). We show here that this functional stimulation is a manifestation of a tight interaction between UL9 protein and ICP8. By using protein-affinity chromatography, we have demonstrated the specific binding of purified UL9 protein to immobilized ICP8 and vice versa. Furthermore, ICP8 is specifically retained by a column on which the C-terminal 37-kDa DNA-binding domain of the UL9 protein was immobilized. The interaction between ICP8 and the DNA-binding domain of the UL9 protein was confirmed by cochromatography of the two proteins. These results suggest that the UL9 protein and ICP8 form a tight complex that functions in origin recognition and unwinding.