Antiviral drugs alone have been unsuccessful in the treatment of Epstein-Barr virus (EBV)-associated malignancies because the virus maintains a latent state of replication in these tumors. In recent years, novel therapeutic approaches are being investigated wherein lytic replication of the virus is induced prior to the use of cytotoxic antiviral drugs. The choice of suitable agents to induce lytic replication has been a critical step in this novel approach. We have previously demonstrated that butyrate derivatives induce a lytic pattern of EBV gene expression in patient-derived EBV-positive lymphoblastoid cell lines and, together with nucleoside analogue ganciclovir, effectively reduce or eliminate tumor growth in humans. Butyrate has drawbacks as a therapeutic agent, however, as constant intravenous infusion is required to achieve detectable plasma levels of this drug. In this study, we investigated whether discontinuous exposure to butyrate is capable of initiating lytic phase gene expression and thymidine kinase induction, and sensitizing EBV-positive lymphoma cells to ganciclovir-mediated cell growth arrest and apoptosis. We demonstrate that multiple daily 6-h exposures of the EBV-positive Burkitt's lymphoma cell line P3HR1 to butyrate induced sustained expression of the EBV lytic phase protein BMRF. Viral thymidine kinase was also induced by intermittent exposure, although to a lower level than with continuous exposure treatment. However, discontinuous exposure to butyrate in combination with ganciclovir induced a similar level of tumor cell growth inhibition as did continuous treatment, as measured by serial enumeration of viable cells, MTT cell proliferation assays and measurement of cellular DNA content. We further demonstrated that those cells which survived initial exposure to butyrate plus ganciclovir remained susceptible to further cycles of combination treatment. These findings suggests that continuous infusion of butyrate may not be necessary for maintaining viral thymidine kinase gene expression and sensitization to antiviral agents in EBV-associated tumors, and that therapeutic regimens which employ more convenient, discontinuous exposure to butyrate may also be effective clinically.