The hot-water extract of Geum japonicum has been shown to exhibit prophylactic and therapeutic anti-herpes simplex virus (HSV) activity in murine infection models. Eugeniin was purified as an anti-HSV compound from the extract and also was isolated from another herbal extract (Syzygium aromaticum) that had exhibited anti-HSV activity in mice. Thus the anti-HSV action of eugeniin was characterized. The effective concentration (5.0 microg/ml) for 50% plaque reduction of eugeniin for wild HSV type 1 (HSV-1) on Vero cells was 13.9-fold lower than its 50% cytotoxic concentration determined by a yield-reduction assay. Eugeniin also inhibited the growth of acyclovir-phosphonoacetic acid-resistant HSV-1, thymidine kinase-deficient HSV-1 and wild HSV type 2. Eugeniin as well as phosphonoacetic acid inhibited viral DNA and late viral protein syntheses in their infected Vero cells, but not cellular protein synthesis at its inhibitory concentrations. Purified HSV-1 DNA polymerase activity was inhibited by eugeniin noncompetitively with respect to dTTP. Its apparent Ki value for euginiin was 8.2- and 5. 8-fold lower than the Ki values of purified human DNA polymerases alpha and beta, respectively. Thus one of the major target sites of inhibitory action of eugeniin is viral DNA synthesis; the inhibitory action for viral DNA polymerase activity was novel compared with anti-HSV nucleoside analogs.