BACKGROUND: Airway smooth muscle contraction causes bronchial constriction and is the main cause of bronchospasm in response to stimulants in asthma patients. In this pilot study, we tested the possibility of using a commercially available neurotoxin-botulinum toxin A (BTX-A)-to reduce bronchial hyperreactivity in dogs. METHODS: Two bronchoscopic sessions were conducted in 6 healthy mongrel dogs. In the first session, BTX-A (concentration 10 U/mL) was injected in small aliquots submucosally in 1 caudal lobe and its subsegments, leaving the other side as control. During the second bronchoscopy conducted 2 weeks later, the airway calibers of the treated and untreated sides were measured in each animal before and after instillation of methacholine in the airways to induce bronchial hyperreactivity (concentration 25 mg/mL). RESULTS: The mean pretreatment diameter was 3.356 (± 1.294) mm and 2.765 (± 0.603) mm in the treated and untreated airways, respectively. After provocation with methacholine, the diameter of the treated airways was 1.985 (± 0.888) mm versus 0.873 (± 0.833) mm in the untreated airways (P=0.000). Local injection of BTX-A in the airway resulted in reduction of bronchial hyperreactivity by 58.6% (P=0.001). There were no complications resulting from the submucosal injection of BTX-A in the airways. CONCLUSIONS: Endobronchial injection of BTX-A reduces bronchial hyperreactivity in the airways of healthy dogs.