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We investigated whether the airways of the lungs from 8 to 10 g fetal pigs (the pseudoglandular phase) have a nerve supply, are functionally innervated and narrow in response to electrical field stimulation and to agonists. Measurements of airway narrowing were made by real-time video imaging of intact isolated bronchial tree freed of associated parenchyma and vasculature. The distal (100 to 300 microns lumen diameter) and terminal (approximately 25 to 50 microns lumen diameter) airways narrowed strongly to acetylcholine and histamine, to within 50 to 80 microns of the base of the epithelial buds. Electrical field stimulation produced rapid narrowing followed by relaxation, and responses were blocked by atropine and tetrodotoxin, indicating a functional cholinergic innervation. Transient periods (3 to 5 min) of spontaneous narrowing were seen in localized regions of the bronchial tree which moved the lung liquid to and fro. An extensive nerve supply to the bronchial tree was seen after immunocytochemical staining of tissue whole mounts with anti-neurofilament. Nerves supplied the distal and terminal airways with fine branches penetrating the smooth muscle of the airway wall. A few fibers extended to the growing edge of the smooth muscle at the very base of the epithelial bud. The smooth muscle was cylindrically arranged around the airway wall all the way to the epithelial bud. We propose that onset of function of smooth muscle and its innervation occurs shortly after differentiation of the smooth muscle at the growing tips of the airways.