The purpose of this study was to analyze the effect of some pharmaceutical excipients when used for mucosal vaccine formulations and to characterize the achieved immune response. After conducting various pharmaceutical evaluations of the formulations, immunokinetic studies were performed in mice, guinea pigs, and rabbits. The kinetics and the characteristics (antibody isotypes, etc.) of the immune response were studied, as well as the induced level of toxin neutralizing IgG antibodies, which are usually used as the only measures of the potency of vaccines. Results in mice show that intranasal vaccination results in a potent and rapid immune response, similar to that seen after subcutaneous immunization. In guinea pigs and rabbits, however, the subcutaneous immunization produced significantly stronger response than did intranasal vaccination. The most promising excipients were found to be either Polysorbate 20 or Cremophor EL in an aqueous mixture together with caprylic/capric glyceride. The results indicate that nontoxic and pharmaceutically acceptable excipients can be used for mucosal vaccination, providing an interesting alternative to parenteral vaccination.