This study combined single and transneuronal labeling to define the origin of midline-crossing vagal fibers projecting to the rat's lungs. Injections of the -subunit of cholera toxin (CT-) into the lungs labeled similar numbers of neuronal somata in the nucleus ambiguus and dorsal motor nucleus of the vagus on both sides of the medulla, even though vagal stimulation increased lung resistance 50% less in the contralateral than in the ipsilateral lung. Unilateral cervical vagotomy prevented CT- labeling of ipsilateral neuronal somata and sensory fibers, indicating that lung-bound vagal fibers undergo decussation only inside the thorax. Injections of CT- and FluoroGold into opposite main stem bronchi double labeled 30% and 11% of all neuronal somata immunoreactive for CT- and FluoroGold, respectively, showing that one single vagal motoneuron can innervate airways on both sides. Injections of pseudorabies virus into the right lung revealed a bilateral network of infected neurons, even after unilateral vagotomy. The latter did not prevent infection of the ipsilateral vagal nuclei. These findings demonstrate that vagal motoneurons that project to the lungs receive contralateral inputs from the airway premotor network and vagal bronchomotor centers.