The cytotoxic T-cell response induced by primary Epstein-Barr virus (EBV) infection and detectable in the blood of infectious mononucleosis (IM) patients shows several unusual features when tested in in vitro assays. Lysis of EBV-transformed target lines occurs with no apparent HLA restriction, and the putative EBV specificity of the response has been seriously questioned. In the present work we show that the primary T-cell response in IM is polyclonal and indeed does contain a virus-specific HLA class I antigen-restricted component, which can be selectively expanded in vitro in the presence of appropriate stimulator cells and IL-2. This allows functional analysis of the virus-specific component of the response in the absence of co-resident reactivities. Studies on blood samples taken from individuals in the acute phase of IM and again post-convalescence suggest that functionally similar populations of HLA class I-restricted cytotoxic T cells are involved in the control of both the primary and persistent phase of EBV infection.