We previously reported that extracellular particles (EPs) composed of premembrane (prM) and envelope (E) proteins were released from cells infected with recombinant vaccinia viruses encoding Japanese encephalitis (JE) virus prM and E genes. In the present study, EPs were evaluated for induction of JE virus-specific antibody and specific T lymphocytes in mice. Six- to 8-week-old male Balb/c mice were inoculated intraperitoneally once or twice (at a 3-week interval) with purified EPs containing 1 microgram of E without adjuvant. Neutralizing antibody was detected and spleen cells proliferated against JE viral antigen 3 weeks after the second immunization with EPs. Neutralizing antibody and JE virus-specific T lymphocytes were also detected 10 months after immunization with EPs containing 2 micrograms of E. Spleen cells obtained from EP-immunized mice and stimulated in vitro with live JE virus, expressed JE virus-specific cytotoxic activity. The cytotoxic activity was reduced by treatment with anti-CD3 antibody and complement. These results indicate that immunization with EPs induces long-lasting specific antibody and memory T cells in mice.