The Epstein-Barr virus (EBV) is characterised by the ability to infect human B lymphocytes in vitro and to transform and immortalise them into lymphoblastoid cell lines (LCLs). EBV strains can be divided into type 1 and type 2, according to the sequence of the viral EBNA-2 protein. Type 1 strains exhibit a superior capacity to efficiently transform B cells and this was mapped to the type 1 EBNA-2 protein. Substitution of the serine residue located in the transactivation domain (TAD) of type 2 EBNA-2 by the equivalent aspartate-442 of type 1 protein was previously shown to confer a type 1-like cell growth phenotype to type 2. In the present study, a stronger ability to transactivate gene expression was demonstrated for type 1 EBNA-2 TAD in comparison to type 2 EBNA-2 and this was determined by aspartate-442. This residue is located next to the interaction site of the transcriptional repressor BS69 with EBNA-2. BS69 inhibits gene transactivation by type 1 and type 2 EBNA-2 but a higher BS69 affinity for type 2 EBNA-2 protein has been shown in this study. Selective EBNA-2 activation of target promoters is also involved in the differential induction of target genes by type 1 and type 2 EBNA-2. Biotinylated oligonucleotide pulldown assays showed that efficient type 1 EBNA-2 binding at the LMP-1 promoter is dependent on the PU.1 and octamer motifs. Greater binding of type 1 EBNA-2 at the differentially regulated promoters, in conjunction with its stronger transactivation ability, conferred by aspartate-442, may determine the higher induction of the LMP-1 and cellular genes required for cell growth proliferation and therefore result in superior B cell transformation by type 1 EBV.