Primary Epstein‐Barr virus (EBV) infection, when manifest as infectious mononucleosis (IM), induces a broad‐ranging and apparently non‐HLA‐restricted cytotoxic response whose nature has not been resolved. In the present experiments the ability to cryo‐preserve IM mononuclear cell preparations, after depletion of CD16⁺ natural killer cells, has allowed detailed analysis of the response on appropriately constructed target cell panels. The results show that IM effector preparations are polyclonal with separate HLA class I antigen‐dependent reactivities against (a) the autologous EBV‐transformed lymphoblastoid cell line (LCL) and (b) particular HLA class I‐mis‐matched LCLs. The autologous LCL‐directed response shows the hallmarks of immunologically specific T cell cytotoxicity; only EBV⁺ B cell blasts are recognized and the interaction can be blocked by monoclonal antibodies to CD3 and CD8 on the effector cell surface and to HLA class I antigens on the target cell. Such findings demonstrate, for the first time, that the primary cytotoxic response to EBV infection includes a virus‐specific HLA‐restricted component like that found in the T cell memory of persistently infected individuals. Separate components of the response are preferentially active against some (but not all) HLA‐mismatched LCLs, the pattern of reactivity being distinct for each individual IM patient and reproducible on repeated testing. Monoclonal antibody blocking experiments show that these HLA‐mismatched interactions also involve CD3 and CD8 antigens on the effector cell and HLA class I antigens on the target cell. Where tested, such lysis affected both EBV⁺ and EBV⁻ B cell blasts from the relevant HLA‐mismatched donors. We postulate that a widespread primary infection of the B cell system by EBV leads to a generalized expansion not just of the virus‐specific response but also of other T cell responses coincidentally active at the time. The unusual activity of IM effector preparations against HLA‐mismatched LCLs arises from fortuitous cross‐recognition of allogeneic cells by immunologically specific cytotoxic T cell clones coincidentally expanded in vivo alongside the EBV‐specific response.