Recombinant Japanese encephalitis (JE) vaccine candidates based on a highly attenuated vaccinia virus (NYVAC-JEV) and a canarypox virus (ALVAC-JEV) were evaluated for their ability to induce specific antibodies and cytotoxic T lymphocytes (CTLs) in mice. Six- to eight-week-old male Balb/c mice that received one or two intraperitoneal inoculations with these JE vaccine candidates at a dose of 1 x 10(7) PFU per mouse produced neutralizing antibody and antibodies to the envelope (E) and nonstructural 1 (NS1) proteins as determined by radioimmunoprecipitation. Immunization with either of these vaccine candidates also induced JE virus-specific T lymphocytes that proliferated in response to stimulation with infectious virus and/or noninfectious viral antigens. Mice maintained detectable levels of neutralizing antibody and JE virus-specific memory T cells for at least 6 months after immunization with NYVAC-JEV and for 4 months after immunization with ALVAC-JEV. Cells induced to proliferate after stimulation with live virus contained specific CD8+ CTLs that lysed primary Balb/c mouse kidney cells infected with JE virus and P815 mastocytoma cells infected with a recombinant vaccinia virus expressing the premembrane (prM), E, and NS1 proteins. These CTLs also lysed P815 cells infected with vaccinia recombinants expressing prM and E, and those expressing E and NS1, but did not lyse P815 cells infected with a recombinant virus expressing only NS1, indicating that the CTLs mainly recognized E, but did not recognize NS1. These results demonstrate that both recombinant JE vaccines, NYVAC-JEV and ALVAC-JEV, induce JE virus-specific antibody and CTLs in mice.