Bacillus subtilis strains expressing tetanus toxin fragment C (TTFC) were tested as vaccine candidates against tetanus in adult mice. Mice received three intranasal (IN) exposures to 10(9) spores or 10(8) vegetative cells of B. subtilis expressing recombinant TTFC. Immunized mice generated protective systemic and mucosal antibodies and survived challenge with 2× LD(100) of tetanus toxin. Isotype analysis of serum antibody indicated a balanced Th1/Th2 response. Lyophilized vaccines stored at 45° C for ≥ 12 months, remained effective. Immunized conventional and SCID mice remained well, and no histological changes in brain or respiratory tract were detected. Lyophilized/reconstituted B. subtilis tetanus vaccines administered IN to mice appear safe, heat-stable, and protective against lethal tetanus challenge.