Unfractionated mononuclear (UM) cells and T cells freshly prepared from the blood of adult donors were co-cultivated in microtest plate wells with progressively lower numbers of cells from the autologous EB-virus-transformed B-cell line. The fresh cells present in co-cultures from EB virus antibody-negative (seronegative) donors regularly facilitated autologous cell line outgrowth, monitored after 4 weeks, whereas outgrowth was markedly inhibited in the corresponding co-cultures from seropositive donors. Larger-scale co-cultures, set up at a ratio of 80–100 fresh UM cells to one autologous virus-transformed B cell, were harvested after 8 to 12 days and the T-cell subpopulation was examined for cytotoxicity both by growth inhibition and by chromium release assays. Cytotoxic T cells were generated exclusively in seropositive donor co-cultures and were strongly active against the autologous virus-transformed cell line without affecting either autologous uninfected B cells or any of a range of EB virus genome-negative target cell lines chosen as sensitive indicators of non-specific cytotoxicity. Recognition of allogeneic EB-virus-transformed cells was restricted to those whose HLA-A and/or B and/or C antigen expression matched that of the effector cells themselves; moreover target cell lysis was specifically inhibited in the presence of monoclonal antibodies binding to these HLA antigens. The results indicate that EB-virus-specific HLA-restricted memory T cells, present in the blood of previously-infected individuals, can be reactivated in vitro using the established autologous virus-transformed cell line as a stimulus. The reactivated cytotoxic cells appear to recognize a virus-induced lymphocyte-detected membrane antigen, LYD-MA, analogous to that first invoked to explain the cytotoxic response to primary EB virus infection observed during infectious mononucleosis.