Epstein-Barr virus (EBV) nuclear antigen (EBNA) 2 (EBNA2) is involved in upregulating the expression of both EBNAs and latency-associated membrane proteins. Transcription of the six EBNA genes, which are expressed in EBV-immortalized primary B cells, arises from one of two promoters, Cp and Wp, located near the left end of the viral genome. Wp is exclusively used to drive EBNA gene transcription during the initial stages of infection in primary B cells; induction of transcription from Cp follows. We previously have mapped an EBNA2-dependent enhancer upstream of Cp (M. Woisetschlaeger et al., Proc. Natl. Acad. Sci. USA 88:3942-3946, 1991) and, more recently, have demonstrated that deletion of this enhancer results in EBV-immortalized lymphoblastoid cell lines (LCLs) that are heavily biased toward the use of Wp to drive transcription of the EBNA genes (L. Yoo et al., J. Virol. 71:9134-9142, 1997). To assess the immortalizing capacity of this mutant EBV and to monitor the early events after infection of primary B cells, B cells isolated from cottontop marmosets were used to generate LCLs immortalized with the Cp EBNA2 enhancer deletion mutant virus. As previously reported, all EBV-infected marmoset LCLs examined could be triggered to produce significant levels of virus. Infection of human B cells with wild-type or Cp EBNA2 enhancer mutant viruses recovered from marmoset B-cell lines demonstrated that (i) the Cp EBNA2 enhancer mutant virus immortalizes primary human B cells nearly as efficiently as wild-type virus and (ii) the Cp EBNA2-dependent enhancer plays an important role in the induction of Cp activity during the early stages of infection. The latter is consistent with the phenotype of LCLs immortalized with the Cp EBNA2 enhancer mutant EBV. Finally, using an established LCL in which EBNA2 function is regulated by -estradiol, we showed that the loss of EBNA2 function results in an ~4-fold decrease in the steady-state levels of Cp-initiated transcripts and a concomitant increase in the steady-state levels of Wp-initiated transcripts. Taken together, these results provide strong evidence that EBNA2 plays an important role in regulating Cp activity. These results also demonstrate that diminished induction of Cp activity does not appear to affect the ability of EBV to immortalize primary B cells in cultures. Finally, as shown here, infection of marmoset B cells with immortalization-competent mutants of EBV provides a convenient reservoir for the production of mutant viruses.