There remains a lack of agreement on the effect of antiviral therapy on herpes simplex virus (HSV) latency and subsequent reactivation. To gain insight into this important issue, a single-cell polymerase chain reaction assay was used to quantify the effects of high-dose acyclovir on latent infection in a mouse model. Treatment with 50 mg/kg of acyclovir every 8 h reduced the number of latently infected neurons by >90% when treatment was begun before 24 h after infection and by 80% and 70% when begun at 48 or 72 h after infection, respectively. The biologic significance of these reductions was evaluated by using a well-established in vivo reactivation model. The number of animals in which virus reactivated was reduced significantly, even when acyclovir therapy was delayed until 72 h after infection, a time when animals had developed lesions. These findings indicate that potent antiviral therapy during early primary HSV infection can reduce the magnitude of the latent infection, such that a significant decrease in reactivation is observed