Cytotoxic T lymphocytes (CTLs) recognising viral antigens are an important host defence mechanism in restricting the proliferation of virus-infected cells. Previously, lymphoblastoid cell lines (LCLs) infected with vaccinia recombinants encoding viral proteins have been used to identify specific CTL epitopes. However, to localise the EBV CTL epitopes encoded by Epstein-Barr virus (EBV) transformants, LCLs are an inappropriate host for vaccinia recombinants. In the present study, an alternative host cell for vaccinia infection is described. Initial studies demonstrated that anti-CD40 stimulated human B cells replicated vaccinia virus and expressed EBV nuclear antigen(s) (EBNA) following infection with recombinant vaccinia encoding the appropriate region of the EBV genome. Recombinant vaccinia-infected anti-CD40 stimulated B cell lines were then used to localise target epitopes for a panel of EBV-specific CTL clones. Most importantly, in vitro stimulation of unfractionated mononuclear cells with recombinant vaccinia-infected anti-CD40 B cells activated a memory CTL response. Based on the vaccinia results, screening of peptides from EBNA6 defined the epitope for the EBNA6-specific CTL clones to the sequence KEHVIQNAFRK. This work clearly demonstrates that anti-CD40 stimulated B cell lines not only provide an efficient tool for localising CTL epitopes but also presents an alternative mechanism of reactivating a memory T cell response to any gene product expressed by recombinant vaccinia.