Several H-2 defined cell lines were examined for their ability to support infection and replication of Japanese encephalitis virus (JEV) before their use in in vitro and in vivo stimulation protocols for generating cytotoxic T lymphocytes (CTLs) against JEV. Among 11 different cell lines tested, two H-2d macrophage tumour lines (P388D1, RAW 264.7), an H-2d hybridoma (Sp2/0), an H-2KkDd neuroblastoma (Neuro 2a), and H-2k fibroblast cell line (L929) were found to support JEV infection and replication. These cell lines were used to generate anti-JEV CTLs by using in vivo immunization followed by in vitro stimulation of BALB/c mice. We observed that not only syngeneic and allogeneic infected cells but also JEV-infected xenogeneic cells could prime BALB/c mice for the generation of JEV-specific CTLs upon subsequent in vitro stimulation of splenocytes with JEV-infected syngeneic cells. Although infected xenogeneic cells were used for immunization, the anti-JEV effectors that were generated lysed infected syngeneic targets but not JEV-infected xenogeneic or allogeneic target cells in a 5 h 51Cr release assay. These anti-JEV effectors recognized syngeneic target cells infected with West Nile virus to a lesser extent and were shown to be Lyt-2.2+ T cells. The results of unlabelled cold target competition studies suggested alterations in the cell surface expression of viral antigenic determinants recognized by these CTLs. We further demonstrate that the JEV-specific CTLs generated could virtually block the release of infectious virus particles from infected P388D1 and Neuro 2a cells in vitro.