Epstein-Barr virus (EBV) transforms resting primary human B lymphocytes into indefinitely proliferating lymphoblastoid cell lines in vitro and is associated with several human malignancies in vivo. Recombinant EBV genetic analyses combined with in vitro B lymphocyte transformation assays demonstrate that latent infection membrane protein 1 (LMP1) is essential for EBV-mediated lymphocyte transformation. LMP1 has no intrinsic enzymatic activity but instead aggregates cellular proteins of the tumor necrosis factor receptor signaling pathway to activate transcription factor NF-kappaB. Mutants rendering LMP1 defective in these protein interactions are impaired in their abilities to activate NF-kappaB in reporter gene assays. Concordantly, EBV recombinants with LMP1 mutations that are compromised for NF-kappaB activation are impaired for growth transformation. Thus, EBV-mediated growth transformation is genetically and biochemically linked to LMP1-mediated activation of NF-kappaB.