The efficacy of a DNA vaccine against western equine encephalitis (WEE) infection in mice was evaluated. The 26S structural region was expressed, in vitro from an internal T7 promoter using a rabbit reticulysate transcription/translation system; and from a CMV promoter after transfection into Vero cell monolayers. The proteins synthesized were reactive with anti-WEE virus (WEEV) antibodies, both in western blot analysis and histochemical staining, respectively. When the DNA vaccine plasmid, pVHX-6, was administered intraepidermally to mice, followed by challenge in a lethal mouse model, the level of protection obtained ranged from 50 to 100% amongst three strains of WEEV. Preliminary results suggest the protective immunity provided by the DNA vaccine appears to be a cell-mediated immune response, as elevated cytotoxic T lymphocyte activity was detected against the E2 protein in a T-cell proliferation assay. The efficacy results suggest a DNA vaccine may be a promising approach against WEE infection.