Three Epstein-Barr virus (EBV) nuclear antigen (EBNA)-encoded oligopeptide epitopes have been mapped, each capable of acting as a recognition determinant for class I-restricted lysis by CD8+ cytotoxic T lymphocytes (CTL). This report shows that each peptide, when presented on an appropriate autologous antigen-presenting cell (APC), also stimulates EBV-specific memory T cells present in peripheral blood mononuclear cell (PBMC) populations to develop in vitro into peptide-specific CTL. These CTL specifically lysed autologous EBV-infected lymphoblastoid cell lines (LCL) and peptide-sensitized uninfected targets. Identical viral oligopeptides could therefore function as recognition determinants for both the induction and commission of class I-restricted specific cytotoxicity. A model system is described in which autologous phytohaemagglutinin (PHA) blasts present exogenous peptide during the stimulation phase. The magnitude of the peptide-specific CTL response was dependent on the concentration of peptide added to the APC and specific lysis was inhibited by anti-class I monoclonal antibody (MoAb) but not anti-class II MoAb. Cultures depleted of CD8+ T cells by cell separation with immunomagnetic beads prior to stimulation invariably failed to generate a peptide-specific CTL response. However, the effect of CD4 depletion on CTL activity was equivocal and indicated that a need for CD4+ T cells as accessory helper cells may depend on the efficiency of the APC to elaborate their own help. This model has advantages in the analysis of events involved in the development of CTL activity in vitro.