Treatment of African green monkey kidney cells with 1 μ/ml of (S)-1-[(3-hydroxy-2-phosphonyl methoxy) propyl] cytosine (HPMPC) inhibited the release of infectious herpes simplex virus type 1 (HSV-1) by more than 90%. Electron microscopic observations of HPMPC-treated monkey kidney cells demonstrated few intracellular or extracellular viral particles. The viral particles seen were without dense cores. In addition, HPMPC blocked cell fusion induced by HSV-1 in monkey kidney cells. Immunoblot analysis showed that HPMPC significantly blocked the expression of HSV-1-specific proteins. Furthermore, HPMPC inhibited the synthesis of viral DNA as determined by in situ hybridization. These results indicate that HPMPC inhibits the replication of HSV by blocking one of the events involved in DNA synthesis. © 1992.