The Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA2) is one of the first EBV-encoded gene products expressed after infection of primary B lymphocytes. EBNA2 is essential for the growth-transforming potential of the virus and it functions as a transcriptional activator of a set of viral and cellular genes. Sequence-specific DNA-binding by EBNA2 has not been demonstrated but the molecule is targeted to specific DNA regions by a cellular protein, RBP-J kappa, which recognizes the GTGGGAA sequence present in the regulatory region of all EBNA2-responsive promoters defined so far. We have determined the contribution of a RBP-J kappa recognition sequence, an adjacent interferon-stimulated response element (ISRE) motif and a PU.1-binding site in the LMP1 regulatory sequence (LRS) to EBNA2-induced transactivation of the promoter by site-directed mutagenesis of LRS-carrying reporter plasmids. EBNA2 responsiveness was reduced by approximately twofold when either or both of the RBP-J kappa-binding and ISRE sequences were mutated. ISRE seemed to function as an EBNA2-independent positive element. On the other hand, mutation of the PU box resulted in a drastic reduction of EBNA2 responsiveness, irrespective of whether the RBP-J kappa site or the ISRE motif was present. A comparative study by deletion mutation identified regions of EBV B95-8 EBNA2 involved in the transactivation of the LMP1 and the EBNA Cp promoters. Two domains of EBNA2 defined by deletion of amino acids 247-337 and 437-476 were found to be important for the activation of both promoters, while two different domains corresponding to residues 4-18 and 118-198 were required solely for the LMP1 promoter. Thus, EBNA2 must activate the LMP1 and Cp promoters by different mechanisms. All deletions involved in transcriptional activation of the two promoters contained regions that are conserved in EBNA2 of B95-8 EBV (type 1), AG876 EBV (type 2) and herpesvirus papio origin.