Immune responses against the protective antigen (PA) of Bacillus anthracis are known to confer immunity against anthrax. We evaluated the efficacy of genetic vaccination with plasmid vectors encoding PA, in protecting mice from a lethal challenge with B. anthracis STI spores. BALB/c and A/J mice were immunized via gene gun inoculation, using eukaryotic expression vectors with different cellular targeting signals for the encoded antigen. The vector pSecTag PA83, encoding the full-length PA protein, has a signal sequence for secretion of the expressed protein. The plasmids pCMV/ER PA83 and pCMV/ER PA63, encoding the full-length and the physiologically active form of PA, respectively, target and retain the expressed antigen in the endoplasmic reticulum of transfected cells. All three plasmids induced PA-specific humoral immune responses, predominantly IgG1 antibodies, in mice. Spleen cells collected from plasmid-vaccinated BALB/c mice produced PA-specific interleukin-4, interleukin-5, and interferon-gamma in vitro. Vaccination with either pSecTag PA83 or pCMV/ER PA83 showed significant protection of A/J mice against infection with B. anthracis STI spores.