Although genetic and biochemical data indicate that the cell protein RBPJkappa is a mediator of EBNA-2 and EBNA-3C effects on transcriptional regulatory elements, the extent of association of these Epstein-Barr virus nuclear proteins with RBPJkappa in transformed B lymphocytes has not been determined. We now report that most of the EBNA-2 and at least 20% of the EBNA-3C coimmunoprecipitated with RBPJkappa from extracts of transformed B lymphocytes that contained most of the cellular EBNA-2 and EBNA3C. Both proteins are associated preferentially with the smaller of the two RBPJkappa isoforms. EBNA-2-RBPJkappa complexes do not contain EBNA-3C, and EBNA-3C-RBPJkappa complexes do not contain EBNA-2. Although EBNA-2 and EBNA-3C are extensively associated with RBPJkappa, a fraction of RBPJkappa appears to be free of EBNAs after repeated immunoprecipitations with anti-EBNA, Epstein-Barr virus-immune, human antibody. Promoters with RBPJkappa sites in their regulatory elements are likely to be differentially regulated by these RBPJkappa-EBNA-2 and RBPJkappa-EBNA-3 complexes.