Japanese encephalitis virus (JEV), which belongs to the family Flaviviridae, causes infection of the central nervous system in humans and equines and stillbirths in swine. In the present report, we constructed and characterized the immune responses conferred by recombinant adenoviruses expressing JEV E epitopes (six amino acid residues 60-68, 327-333, 337-345, 373-399, 397-403 and 436-445 in E, designated TEP). Seven groups (n = 10) of female BALB/c mice received intramuscular (IM) or oral immunization with the recombinant adenoviruses twice at 2-week intervals. Intramuscular immunization of mice with rAd-TEP generated greater titers of anti-JEV antibodies and JEV neutralizing activity than in animals with oral injection. It statistically significant differences were found in anti-JEV antibody titers and JEV neutralizing activity induced by IM immunization with rAd-TEP at a dose of 1 x 10(8.0)TCID50 when compared with the doses tested (3 x 10(7.0) and 1 x 10(7.0)TCID50) IM inoculation of rAd-TEP. Splenocytes from mice immunized intramuscularly with rAd-TEP secreted the largest amounts of interferon-gamma and interleukin-2 and moderate amounts of interleukin-4 in the presence of JEV. It demonstrates that IM immunization with rAd-TEP induced the highest level of cell-mediated immune responses and the higher level of JEV-specific humoral immune responses than oral immunization. Then we further evaluated the protective efficacy of the recombinants in swine. All swine were protected from viral challenge with IM rAd-TEP at 1 x 10(10.0)TCID50, even though the neutralizing antibody titers were lower than those in the group inoculated with inactivated vaccine. Our findings indicate that rAd-TEP might be an attractive candidate vaccines for preventing JEV infection.