Transcutaneous immunization is a novel strategy for immunization employing topical application of antigen and adjuvant to the skin surface and resulting in detectable antigen/adjuvant specific IgG in plasma and mucosal secretions. In this study we show that transcutaneous immunization with cholera toxin (CT) as an adjuvant can be used in several inbred mouse strains with varying H-2 major histocompatibility complex genes (C57BL/6 (H-2^b), BALB/c (H-2^d), and C3H (H-2^k)). Although the primary anti-CT antibody responses reflected previously described MHC restriction patterns for this protein, the differences were overcome after two booster immunizations. Potent antibody responses against hen egg lysozyme and/or diphtheria toxoid were observed using CT as adjuvant. We also demonstrate that the unshaved dorsal or ventral surface of the ear can be effectively used for transcutaneous immunization and that gentle swabbing with alcohol increases the magnitude of the host immune response. Together these data further our understanding of the principles governing this new platform technology and support its integration into novel and existing human vaccine strategies.