Nicotine acting centrally increases bronchomotor tone and airway secretion, suggesting that airway-related vagal preganglionic neurons (AVPNs) within the rostral nucleus ambiguus (rNA) express nicotinic acetylcholine receptors (nAChRs). In the present study, we examined the three main functionally characterized subtypes of nAChRs in the CNS, the alpha7 homomeric and alpha4beta2 heteromeric receptors. First, we characterized the expression of these subunits at the message (mRNA) and protein levels in brain tissues taken from the rNA region, the site where AVPNs are located. In addition, double labeling fluorescent immunohistochemistry and confocal laser microscopy were used to define the presence of alpha7, alpha4, and beta2 nAChRs on AVPNs that were retrogradely labeled with cholera toxin beta subunit (CTb), injected into the upper lung lobe (n=4) or extrathoracic trachea (n=4). Our results revealed expression of all three studied subunits at mRNA and protein levels within the rNA region. Furthermore, virtually all identified AVPNs innervating intrapulmonary airways express alpha7 and alpha4 nAChR subunits. Similarly, a majority of labeled AVPNs projecting to extrathoracic trachea contain alpha7 and beta2 subunits, but less than half of them show detectable alpha4 nAChR traits. These results suggest that AVPNs express three major nAChR subunits (alpha7, alpha4, and beta2) that could assemble into functional homologous or heterologous pentameric receptors, mediating fast and sustained nicotinic effects on cholinergic outflow to the airways.