The Epstein-Barr virus (EBV) proteins EBNA1, EBNA2, EBNA3A, EBNA3C, LMP1 and EBNA-LP are essential for the in vitro immortalization of primary B lymphocytes by EBV. EBNA2 is a transcriptional activator of viral and cellular genes. Both EBNA3A and EBNA3C have been shown to specifically inhibit EBNA2-activated transcription by direct interaction with RBP-Jkappa, a cellular DNA-binding factor known to recruit EBNA2 to EBNA2-responsive genes. This interaction interferes with the binding of RBP-Jkappa to DNA in vitro, and this is probably the mechanism by which EBNA3A and EBNA3C repress EBNA2-activated transcription in vivo. EBNA3A and EBNA3C also directly repress transcription when tethered to a promoter via the DNA-binding domain of the yeast Gal4 protein. As RBP-Jkappa has been previously shown to be a repressor in mammalian cells, this repression could be due to the recruitment of RBP-Jkappa by Gal4-EBNA3A and 3C. In this study, we have precisely mapped the domain of EBNA3A involved in the interaction with RBP-Jkappa and we have shown that interaction with RBP-Jkappa is not required for the Gal4-EBNA3A-mediated repression. Furthermore, we have characterized in EBNA3A a domain of 143 amino acids which is necessary and sufficient for EBNA3A-dependent repression.