In order to develop an ideal nontoxic mucosal adjuvant, we constructed two cholera toxin (CT) mutants (S61F and E112K) by site-directed mutagenesis. Wild type CT induced morphological changes in Chinese hamster ovary (CHO) cells at 1.0 pg/ml, while neither mutant affected cells even at concentrations of 1.0 μg/ml. Furthermore, these mutants did not induce intracellular cAMP production in CHO cells. C57BL/6 mice were subcutaneously immunized with 100 μg of ovalbumin (OVA) and different doses of mutant CT (1 or 10 μg). As positive controls, 1 μg of CT was used together with OVA. Mice were immunized on days 1 and 14 and serum anti-OVA responses and antibody producing cells were determined one week after the last immunization. Mice immunized with OVA alone showed no significant antibodies to OVA, while mice immunized with OVA and 10 μg of mutant CT showed high levels of antigen-specific IgG1 but not IgG2a responses. These serum responses were comparable to positive controls. These findings were further confirmed by antigen-specific ELISPOT assay. Our results demonstrated not only that adjuvanticity of CT is dissociated from cAMP induction but also that these CT mutants could be candidates for a nontoxic mucosal adjuvant. We are currently examining the ADP-ribosylation activity and in vivo toxicity (rabbit ileal loop test), and mucosal adjuvanticity of the CT mutants.