The intentional release of anthrax in 2001 highlighted the need for vaccines for anthrax and other potential pathogens that could be used as biological weapons. As a result, recent trends in vaccine studies have focused on development of subunit vaccines, but they often require safe and effective adjuvants. Adjuvants are critical components of subunit vaccines as they direct antigen-specific immune responses and affect the immune outcome. For our studies, we used recombinant protective antigen (rPA) from Bacillus anthracis and recombinant fusion protein, F1-V of Yersinia pestis as a model for biodefense antigens. We compared four different adjuvants, LT(R192G), CpG ODN, MPL ^®TDM, and alum, for their ability to affect the magnitude, distribution, and duration of antibody responses against rPA or F1-V in a murine model. In addition, three different routes of immunization—intranasal (IN), transcutaneous (TC), and subcutaneous (SC), were compared with each antigen and adjuvant. Since aerosol exposure of biological warfare agents is of primary concern, both serum and bronchioalveolar lavage (BAL) were analyzed for antigen-specific antibody responses. Additionally, we evaluated synergy or interference when adjuvants were administered SC in combination for their ability to induce F1-V specific immune responses. The most significant findings of these studies are that (1) the adjuvant influences the Type 1/Type 2 balance of the immune response in both the serum and BAL, (2) mucosal immunization is not necessary to obtain rPA or F1-V-specific BAL responses, (3) non-traditional adjuvants such as LT(R192G) work when delivered SC, (4) the route of immunization affects the magnitude of the immune response, (5) rPA and F1-V are immunogenic by some routes even in the absence of an exogenously applied adjuvant, (6) co-administration of LT(R192G) and CpG ODN with F1-V resulted in additive serum, BAL, and cytokine responses when compared to either adjuvant alone, (7) alum suppresses Type 1 immune response in the presence of either LT(R192G) or CpG ODN, and (8) in some vaccine formulations, the adjuvant dose can be decreased when used in combination with another adjuvant in order to achieve superior antigen-specific immune responses.