Latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) is a transforming protein that affects multiple cell signaling pathways and contributes to EBV-associated oncogenesis. LMP1 can be expressed in some states of EBV latency, and significant induction of full-length LMP1 is also observed frequently during virus reactivation into the lytic cycle. It is still unknown how LMP1 expression is regulated during the lytic stage and whether any EBV lytic protein is involved in the induction of LMP1. In this study, we first identified that LMP1 expression is associated with the spontaneous virus reactivation in EBV-infected 293 cells and that its expression is a downstream event of the lytic cycle. We further found that LMP1 can be induced by ectopic expression of Rta, an EBV immediate-early lytic protein. The Rta-mediated LMP1 induction is independent of another immediate-early protein, Zta. Northern blotting and reverse transcription-PCR analysis revealed that Rta upregulates LMP1 at the RNA level. Reporter gene assays further demonstrated that Rta activates both the proximal and distal promoters of the LMP1 gene in EBV-negative cells. Both the amino and carboxyl termini of the Rta protein are required for the induction of LMP1. In addition, Rta transactivates LMP1 not only in epithelial cells but also in B-lymphoid cells. This study reveals a new mechanism to upregulate LMP1 expression, expanding the knowledge of LMP1 regulation in the EBV life cycle. Considering an equivalent case of Kaposi's sarcoma-associated herpesvirus, induction of a transforming membrane protein by a key lytic transactivator during virus reactivation is likely to be a conserved event for gammaherpesviruses.