The ability of Epstein-Barr virus (EBV) latent infection nuclear protein EBNA3C to activate transcription of two EBNA2-responsive genes and to inhibit EBNA2 activation of transcription in transient-transfection assays appears to be due to its ability to interact with RBPJkappa, a cell protein that links EBNA2 to its response elements. We now show that EBNA3A and EBNA3B expressed in non-EBV-infected Burkitt tumor lymphoblasts are similar to EBNA3C in binding to glutathione S-transferase-RBPJkappa in vitro and in coimmunoprecipitating from cell lysates with antibody to RBPJkappa. EBNA3A and EBNA3B can also inhibit the interaction of RBPJkappa with cognate DNA in vitro. Although EBNA3 open reading frames are each close to 1,000 codons long, EBNA3A amino acids 1 to 138, EBNA3B amino acids 1 to 311, and EBNA3C amino acids 1 to 183 are sufficient for RBPJkappa interaction, while EBNA3B amino acids I to 109 have less or no binding. The RBPJkappa interacting domains overlap with the most highly conserved domain (amino acids 90 to 320) among the EBNA3 proteins. Thus, the EBNA3 gene family appears to have evolved to differentially regulate promoters with RBPJkappa binding sites. EBNA2, EBNA3A, and EBNA3C are important in EBV transformation of primary human B lymphocytes. Their interaction with RBPJkappa links EBV transformation to the notch signaling pathway and the effects of activated notch in T-cell leukemogenesis.