Epstein-Barr virus nuclear antigen 2 (EBNA2) is essential for transformation through activation of viral and cellular genes. Within 487 residues, EBNA2 contains six lysine (K) residues (positions 335, 357, 359, 363, 366 and 480), which were mutated to arginine (R) residues, either individually or in combination, and tested for subcellular localization, mobility by SDS-PAGE and transactivation of three promoters. All mutants featuring the K(480)R mutation within the nuclear localization signal were partially cytoplasmic with a reduced level of transactivation of the latent membrane protein 1 (LMP1) promoter (-327 to +40). The K(366)R mutation also showed a decrease in transactivation of a promoter consisting only of 12 recombination signal-binding protein-Jkappa-binding sites, while all mutants with the K(335)R exchange showed a markedly elevated transactivation with the -327 to +40 construct and all mutants showed slightly reduced transactivation with a -634 to +40 LMP1 promoter. None of the mutants exhibited altered migration in SDS-PAGE, excluding secondary modification, i.e. through SUMO-like proteins.