Side effects to botulinum antitoxins, including anaphylaxis and serum sickness, are common. This is due to the immunogenicity of the antitoxin, which can be measured by the production of anti-immunoglobulin antibodies. An ideal botulinum antitoxin would elicit a minimal production of anti-immunoglobulin antibodies from a patient, aiding its safety. To investigate the immunogenicity of different immunoglobulin fragments, whole IgG, F(ab')2 and Fab botulinum antitoxins were administered to mice by either the intravenous or intramuscular route. The production of anti-immunoglobulin antibodies was measured over time after a single dose of antitoxin, and the anti-immunoglobulin antibodies isotyped. When administered by the intramuscular route, Fab showed significantly lower immunogenicity than IgG, while F(ab')2 had an immunogenicity that was intermediate between the two. When administered by the intravenous route there was no significant difference in immunogenicity between IgG and F(ab')2 antitoxins, although Fab antitoxin had a significantly lower immunogenicity than either IgG or F(ab')2. IgG antitoxin was significantly more immunogenic by the intramuscular route than by the intravenous route. Sheep IgG had a lower immunogenicity than goat IgG in mice. There was no significant difference in immunogenicity between the two dosing routes for either F(ab')2 or Fab antitoxin. The anti-antibodies were predominantly IgG1, suggesting a strong Th2 bias to the anti-antibody response. In all cases, Fab represents the least immunogenic form of antitoxin.