Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus that persistently infects 85% of the adult population worldwide. In this report, we examine the proliferative response and cytokine secretion profile of CD4(+) T lymphocytes from a panel of unrelated EBV-positive donors against two EBV latent antigens, EBNA1 and EBNA3C. Substantial proliferative responses by CD4(+) lymphocytes were demonstrated to both antigens in multiple, randomly selected donors. Surprisingly, we observed a striking and consistent difference in cytokine response to EBNA1 and EBNA3C. EBNA1-specific CD4(+) T lymphocytes from multiple unrelated donors preferentially produced type 2-like cytokines in response to antigenic stimulation, while the response to EBNA3C was a characteristic type 1 response. The implications of these findings for EBV persistence and the development of EBV-associated malignancies are discussed.