Hirame rhabdovirus (HIRRV) is an important virus of cultured flounder (Paralichthys olivaceus). We tested the protective immunogenicity of DNA-based vaccines against this virus. Genes encoding the nucleocapsid protein (N) and the C-terminal half of the glycoprotein (G) were amplified by RT-PCR and separately cloned into the eukaryotic expression vector pcDNA 3.1. The G protein expressed by transfected cells was detected by western blot analysis. PCR analyses demonstrated the presence of injected plasmids in fish muscle tissue at 14 days post injection. Immunocytochemistry of muscle tissue injected with the plasmid DNA showed expression of the target protein in myofibrils and sarcoplasm. Flounder fry with an average weight of 3 g were injected with 5 microg of plasmid DNA and challenged at 21 days after immunization. Fish injected with vector DNA or PBS showed >95% cumulative mortality by 16 days after inoculation with the virus. In contrast, fish injected with plasmids containing the N gene, G gene, or N + G gene mixture showed 70, 5, and 2.5% cumulative mortality, respectively. These results show that the G gene is effective for the induction of protective immunity against HIRRV infection in injected fish.