The Epstein-Barr virus (EBV) protein EBNA2, which is essential for the immortalization of human primary B cells by EBV, acts as a transcriptional activator of cellular and viral genes. Specific responsive elements have been characterized in several of the promoters activated by EBNA2. They all share the core sequence GTGGGAA. EBNA2 does not, however, bind to these sequences directly, but appears to be targeted to them by a cellular protein. A similar core sequence has recently been identified as a high-affinity binding site for the human recombination signal sequence binding protein RBP-J kappa. Here we provide evidence that RBP-J kappa binds to specific sequences in EBNA2-responsive elements. Our results also demonstrate that RBP-J kappa makes direct physical contact with EBNA2 in solution and recruits EBNA2 to its cognate DNA sequences, suggesting that RBP-J kappa may mediate EBNA2 transactivation of both cellular and viral genes.