Introduction: In some patients undergoing continuous botulinum toxin treatment, the treatment response reduces, although the frequency is low. This may be because of the production of toxin-neutralizing antibody. To examine the efficacy of treatment with the botulinum neurotoxin subtype A2 (A2NTX) in patients in whom treatment against botulinum neurotoxin subtype A1 (A1NTX) has become less effective, we compared the antigenicity of A1NTX with that of A2NTX using rats immunized with botulinum neurotoxin complex subtype A1 (A1LL).Methods: We prepared a toxoid of A1LL, and subcutaneously administered it at 10μg to rats 3 times at 2-week intervals. These rats were divided into 2 groups so that the neutralizing antibody titer was similar. Amounts of A1NTX and A2NTX showing equivalent activities were administered into the gastrocnemius of rats. We measured a decrease in the CMAP amplitude the day after administration, and evaluated residual toxin activity after neutralization with antibody.Results and Discussion: In the A1NTX-treated group, the toxin was completely neutralized by the antibody. There was no decrease in the CMAP amplitude. In the A2NTX-treated group, the CMAP amplitude was significantly decreased. This suggests the usefulness of treatment with A2NTX in patients in whom conventional A1 strain-derived botulinum toxin preparations induced antibody production.