Epstein-Barr virus (EBV)-specific cytotoxic T lymphocyte (CTL) responses were evaluated in 9 children aged 20-35 months. Autologous EBV-transformed B lymphoblastoid cell lines were used to restimulate EBV-specific memory CTL precursors in vitro. Recognition of individual EBV gene products by bulk CTL lines was evaluated by combining CTL lines with B cell blasts infected with recombinant vaccinia constructs expressing single latent genes. CTL lines from all 9 children recognized one or more EBV latent gene products. All children demonstrated CTL responses against one or more EBV nuclear antigen 3 proteins (EBNA3A, 3B, 3C), and EBNA3C was recognized most frequently. The striking similarity between EBV-specific CTL responses described here in young children and those reported for adults suggests that the EBNA3 family of proteins and latent membrane protein 2A should be considered for inclusion in candidate EBV vaccines.