The hallmark of infection by human herpesviruses, life-long persistence in the host, is unaffected by current antiviral therapies effective against replication of virus. In vitro studies indicated that low concentrations of the ribonucleotide reductase inhibitor, hydroxyurea, completely eliminated Epstein-Barr virus (EBV) episomes from latently infected Burkitt's lymphoma (BL) cell subsets, providing the first example of chemotherapeutic eradication of a latent herpesvirus from any cell population. Unlike parental EBV-positive BL cells, virus-free cell progeny from one treated cell line no longer exhibited the malignant phenotype in tumorigenicity assays. Hydroxyurea-treated primary B lymphocytes immortalized by EBV ceased to proliferate as episomes were lost. The altered growth phenotype of both BL cells and immortalized primary B cells suggests that latent EBV is an appropriate and accessible therapeutic target for treatment of some EBV-induced lymphoproliferations.