A recombinant baculovirus overexpressing the herpes simplex virus type 1 (HSV-1) origin binding protein, encoded by the UL9 gene, was constructed. The purified recombinant protein has DNA-dependent nucleoside triphosphatase activity similar to the enzyme isolated from mammalian cells. Optimal nucleoside triphosphatase activity requires low salt (< 50 mM), 2-3 mM Mg2+, alkaline pH (8.3-9.5), high temperature (45 degrees C), and a single-stranded DNA coeffector containing minimal secondary structure. Enzymatic activity is subject to product inhibition, and there appears to be a single nucleotide binding site. The minimal length of single-stranded DNA that elicits enzymatic activity is 14 nucleotides, and activity increases as the length is increased. Saturation for various single-stranded DNA coeffectors is about 10 microM in nucleotide, but the maximum velocity is reduced 2-3-fold for coeffectors containing secondary structure. The HSV-1-encoded single-stranded DNA-binding protein ICP8 specifically stimulates the DNA-dependent nucleoside triphosphatase activity. The kinetics of nucleoside triphosphate hydrolysis exhibit a substantial lag period which can be shortened, but not eliminated, by reduced secondary structure in the DNA coeffector or by increased temperature.