CR2 (CD21), the EBV receptor, was detected on three of four CD4-positive cell lines by indirect fluorescent labeling, and its corresponding mRNA was found by use of the reverse transcription-based polymerase chain reaction. To determine whether CR2 on CD4-positive cells was functional, their ability to be infected by EBV was analyzed. EBV DNA, EBV nuclear antigen 2 (EBNA-2A), and EBV-encoded small RNA (EBER1) transcripts could be detected in CR2-expressing CD4-positive cells following infection by the B95.8 strain of EBV. Analysis of the terminal region showed the EBV genome remained linear following infection, and copy number decreased with time. Since CD4-positive cell lines are targets for HIV-1 infection, the effects of EBV infection on HIV-1 expression were analyzed. HIV-1 replication was upregulated when CD4-positive cells were coinfected with EBV strain B95.8 but not P3HR-1K. These results suggested that EBNA-2 is involved in upregulation of HIV-1 expression in T lymphoblastoid cell lines. To test this hypothesis an EBNA-2-expression vector was transfected into T lymphoblastoid cell lines and HIV-1 expression measured. First, trans-activation of HIV-1 long terminal repeat (LTR) by Tat was enhanced by EBNA-2 type 1 expression. trans-Activation of the HIV-1 LTR by Tat was also enhanced when CD4-positive cells were infected by EBV (strain B95.8) encoding an intact EBNA-2, but not by P3HR-1K with a deleted EBNA-2. In addition, CD4-positive cell clones stably expressing EBNA-2 supported enhanced HIV-1 replication as measured by accumulation of reverse transcriptase activity and syncytium induction. This provides direct evidence that EBV infection can enhance HIV-1 replication in T cells. Whether this in vitro phenomenon contributes to disease progression in vivo remains to be determined.