Acyclovir [9-(2-hydroxyethoxymethyl)guanine] inhibits Epstein-Barr virus (EBV) replication in lymphoblastoid cells at concentrations nontoxic to cellular growth. The mode of action of the drug against EBV differs from the mechanism described in herpes simplex virus systems. Due to the absence of virus-specified thymidine kinase, the drug is poorly phosphorylated in EBV-infected cells. The extent of monophosphorylation is similar both in mock-infected and EBV-infected cells. Despite weak phosphorylation of the drug, the replication of linear EBV DNA is inhibited due to exquisite sensitivity of the viral DNA polymerase. Activation of acyclovir does not require phosphorylation by virus-specified thymidine kinase, inhibition of different herpes-group viruses depends on three variable factors: degree of phosphorylation, cellular metabolism of the drug, and degree of sensitivity of the viral polymerase. Interaction of acyclovir-triphosphate with EBV DNA polymerase is reversible. Cells infected with EBV and treated with acyclovir resume virus replication following removal of the drug even after long exposure. Acyclovir inhibits replication of linear genomes and stops production of virus, but has no effect on latent cellular infection. These results lead us to predict that acyclovir will suppress, but not cure, EBV infection.