Relatively little is known about the biochemical mechanisms through which the Epstein-Barr virus latent infection integral membrane protein 1 (LMP1) transmembrane domains cause constitutive LMP1 aggregation and continuous cytoplasmic C terminus-mediated signal transduction. We now evaluate the role of the three consecutive LMP1 hydrophobic transmembrane pairs, transmembrane domains (TM)1-2, TM3-4, and TM5-6, in intermolecular aggregation and NF-kappaB activation. LMP1TM1-2 enabled approximately 40% of wild-type LMP1 cytoplasmic domain-mediated NF-kappaB activation, whereas TM3-4 or TM5-6 assayed in parallel had almost no effect independent of LMP1TM1-2. Alanine mutagenesis of conserved residues in LMP1TM1-2 identified FWLY(38-41) to be critical for LMP1TM1-2 intermolecular association with LMP1TM3-6. Further, in contrast to wild-type LMP1, LMP1 with FWLY(38-41) mutated to AALA(38-41) did not (i). significantly partition to lipid Rafts or Barges and effectively intermolecularly associate, (ii). enable cytoplasmic C terminus engagement of tumor necrosis factor receptor-associated factor 3, (iii). activate NF-kappaB, and thereby (iv). induce tumor necrosis factor receptor-associated factor 1 expression. Other LMP1 intermolecular associations were observed that involved LMP1TM1-2/LMP1TM1-2 or LMP1TM3-4/LMP1TM3-6 interactions; these probably also contribute to LMP1 aggregation. Because FWLY(38-41) was essential for LMP1-mediated signal transduction, and LMP1 activation of NF-kappaB is essential for proliferating B lymphocyte survival, inhibition of LMP1FWLY(41)-mediated LMP1/LMP1 intermolecular interactions is an attractive therapeutic target.