Epstein-Barr virus (EBV) is the aetiological agent of infectious mononucleosis (IM) which is a common sequel to primary EBV infection. Thereafter, the virus is maintained as a lifetime latent infection. Although the proteins expressed during the latent EBV infection provide a rich source of immunogenic epitopes, very little is known about cytotoxic T lymphocyte (CTL) control of primary EBV infection. The present report is based on an analysis of CTL clones derived from a patient suffering from acute IM. An intriguing feature of six CTL clones that displayed an HLA-restricted pattern of cell lysis was their initial coexpression of the T cell markers CD3, CD4, and CD8. Detailed analysis of one of these clones, which was restricted through the class II MHC antigen DR2, revealed reactivity with an epitope within the EBV lytic cycle early antigen, BHRF-1, which corresponds to the C-terminal region of the protein (AGLTLSLLVICSYLFISRG) (residues 171-189). There have been no previously published reports describing a CTL response during acute IM directed against an EBV lytic antigen. Interestingly, the coexpression of CD4 and CD8 by these CTLs during acute IM suggests that CD3+CD4+CD8+ cortical thymocytic precursor cells are recruited in order to overcome the EBV infection.