We applied two complementary retrograde labeling techniques to characterize the organization of the brain stem neuronal network responsible for the vagal innervation of the trachealis muscle in developing sheep. Single neuronal labeling produced by injections of the beta-subunit of cholera toxin into the muscle in newborn lambs showed that airway vagal preganglionic neurons are located exclusively in the nucleus ambiguous and nucleus of the solitary tract. Transneuronal labeling produced by similar injections of the Bartha strain of the pseudorabies virus in sheep fetuses demonstrated that these airway vagal preganglionic neurons receive inputs from a small number of neurons in brain stem areas known to participate in premotor control of the respiratory muscles (ventral respiratory group), chemoreception (nucleus of the solitary tract and ventral medullary surface), and cardiovascular and respiratory regulation (raphe nuclei, ventrolateral medulla, and noradrenergic groups of the medulla and pons). We conclude that the vagal preganglionic neurons that project to airway smooth muscle are already integrated in the control of breathing before birth in sheep.