As the indications and duration of treatment of botulinum toxin type A (BoNT-A) increase, so do reports of patients who fail therapy after initially responding well. Although a loss of efficacy is commonly thought to be associated with neutralizing antibodies (NAbs), this relationship is not strongly correlated, and other factors may play a significant role. To explore this issue, we evaluated levels of NAbs in a large selected cohort of secondary nonresponders to BoNT-A using the highly sensitive mouse phrenic nerve-hemidiaphragm assay. Serum samples from 503 patients treated with BoNT-A who had a variety of diagnoses were tested for the presence of NAbs. Fewer than half of the patients (n = 224, 44.5%) were found to be NAb-positive, indicating that in more than half of the secondary nonresponders, lack of efficacy is not due to NAb formation. The proportion of secondary nonresponders with NAbs was greater for higher dose indications (focal spasticity and spasmodic torticollis) than for lower dose indications (blepharospasm and hemifacial spasm) and increased with shorter injection intervals. Neutralizing antibody development was independent of the commercial preparation used. Our results indicate that although NAb formation does play a role in secondary treatment failure with BoNT-A, this is not the cause in all patients, and the influence of other factors needs to be investigated. Gaining a better understanding of the underlying mechanisms for secondary treatment failure may help in the prediction, diagnosis, management, and prevention of this problem.