With the use of a high-throughput biochemical DNA helicase assay as a screen, T157602, a 2-amino thiazole compound, was identified as a specific inhibitor of herpes simplex virus (HSV) DNA replication. T157602 inhibited reversibly the helicase activity of the HSV UL5-UL8-UL52 (UL5/8/52) helicase-primase complex with an IC₅₀ (concentration of compound that yields 50% inhibition) of 5 µM. T157602 inhibited specifically the UL5/8/52 helicase and not several other helicases. The primase activity of the UL5/8/52 complex was also inhibited by T157602 (IC₅₀ = 20 µM). T157602 inhibited HSV growth in a one-step viral growth assay (IC₉₀ = 3 µM), and plaque formation was completely prevented at concentrations of 25 to 50 µM T157602. Vero, human foreskin fibroblast (HFF), and Jurkat cells could be propagated in the presence of T157602 at concentrations exceeding 100 µM with no obvious cytotoxic effects, indicating that the window between antiviral activity and cellular toxicity is at least 33-fold. Seven independently derived T157602-resistant mutant viruses (four HSV type 2 and three HSV type 1) carried single base pair mutations in the UL5 that resulted in single amino acid changes in the UL5 protein. Marker rescue experiments demonstrated that the UL5 gene from T157602-resistant viruses conferred resistance to T157602-sensitive wild-type viruses. Recombinant UL5/8/52 helicase-primase complex purified from baculoviruses expressing mutant UL5 protein showed complete resistance to T157602 in the in vitro helicase assay. T157602 and its analogs represent a novel class of specific and reversible anti-HSV agents eliciting their inhibitory effects on HSV replication by interacting with the UL5 helicase.