A large cumulative dose of botulinum toxin type A (BoNT-A), frequent injections, a short interval between treatment cycles, and a long duration of treatment have all been suggested, but not confirmed, to be associated with a high incidence of neutralizing antibodies to the neurotoxin. The aim of this study was to investigate whether these variables predispose to BoNT-A neutralizing antibody formation. A mouse protection (neutralization) bioassay was used for the detection of BoNT-A antibodies in 17 patients who received large doses of BoNT-A, over at least 10 consecutive treatment cycles or for 5 years or more. BoNT-A antibodies were not detected in any of the study patients. The study findings did not confirm an association between the above-mentioned variables and BoNT-A antibody formation.