This study demonstrated viral antigen production in muscle tissues following inoculation with DNA vaccines and examined its relation to antibody induction in mice using the flavivirus system. To achieve detectable levels of antigen production, we used a needle-free jet injector and examined 10% homogenate of quadriceps muscle for viral antigens in a sandwich enzyme-linked immunosorbent assay. We compared DNA vaccines against dengue type 1 (designated pcD1ME), dengue type 2 (pcD2ME) and Japanese encephalitis (pcJEME). The amounts of viral envelope (E) antigen contained in muscle homogenate 1, 2, 3 and 4 days following inoculation with 50 microg of pcJEME were 1.1, 1.0, 0.3 and <0.1 ng/ml, respectively. Muscles from pcD2ME- and pcD1ME-inoculated mice did not contain detectable levels of E antigen (<0.1 ng/ml) during 4 days following inoculation. The E amounts released from Vero cells transfected with DNAs were in the order pcJEME>pcD2ME>pcD1ME. Levels of neutralizing antibody induced by two immunizations with 100 microg of each DNA vaccine using needle-free or normal needle/syringe injection systems also were in the order pcJEME>pcD2ME>pcD1ME, 2-11 weeks after the first immunization. However, the difference in antibody levels among three DNA vaccines 14-18 weeks after immunization was smaller than that in the early phase of immunization. These results provide fundamental information useful for developing combination DNA vaccines, such as a dengue tetravalent DNA vaccine, which require adjustment of immunogenicity of each component.