The discovery of EBV in certain T cell malignancies and the expression of the EBV receptor, CR2/CD21, on a population of immature thymocytes, T lymphoblastoid cell lines, and childhood acute T lymphoblastic leukemia cells suggested that EBV-receptor interactions on T cells may be of importance. We have shown that, within the thymus, a population of large, immature cells expresses CD21. EBV altered the activation responses of immature thymocytes in vitro. Triggering through CD2 is mitogenic for mature, but not immature, T cells. However, during infection by EBV, ligation of CD2 caused thymocytes to proliferate in the absence of exogenous cytokines. This function was a result of the interaction of EBV with its receptor, CD21, but was caused by infection rather than surface signaling, because neither specific mAb nor the P3HR-1 strain of virus mimicked the effect of B95-8. Immature thymocytes were infected by EBV, as determined by the internalization of the viral genome and its transcriptional activity. Consistent with the activity of B95-8, EBNA-2 transcripts were identified within infected thymocyte populations. In addition, components of the viral replicative pathway were expressed during infection of thymocytes. These components included transcription of BZLF-1, an early gene that characterizes EBV-infected B cells after disruption of latency. A second transcript was identified as encoding the recently characterized RAZ, which also is associated with replicative infection. The consequences of EBV infection of T cells at an early stage of differentiation may lead to failure of normal T cell repertoire development, autoimmunity, or malignancy.