Nasopharyngeal carcinoma, Kaposi's sarcoma, and B-cell lymphomas are human malignancies associated with gammaherpesvirus infections. Members of this virus family are characterized by their ability to establish latent infections in lymphocytes. The latent viral genome expresses very few gene products. The infected cells are therefore poorly recognized by the host immune system, allowing the virus to persist for long periods of time. We sought to identify the cell-specific factors that allow these viruses to redirect their life cycle from productive replication to latency. We find that the cellular transcription factor NF-kappaB can regulate this process. Epithelial cells and fibroblasts support active (lytic) gammaherpesvirus replication and have low NF-kappaB activity. However, overexpression of NF-kappaB in these cells inhibits the replication of the gammaherpesvirus murine herpesvirus 68 (MHV68). In addition, overexpression of NF-kappaB inhibits the activation of lytic promoters from MHV68 and human gammaherpesviruses Kaposi's sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV). In lymphocytes latently infected with KSHV or EBV, the level of NF-kappaB activity is high, and treatment of these cells with an NF-kappaB inhibitor leads to lytic protein synthesis consistent with virus reactivation. These results suggest that high levels of NF-kappaB can inhibit gammaherpesvirus lytic replication and may therefore contribute to the establishment and maintenance of viral latency in lymphocytes. They also suggest that NF-kappaB may be a novel target for the disruption of virus latency and therefore the treatment of gammaherpesvirus-related malignancies.