This study examined the possibility that vasoactive intestinal peptide (VIP)‐ and substance P (SP)‐containing nerve fibers in bronchial smooth muscle, glands, epithelium, and blood vessels originate from neurons of airway ganglia. Explants of airway walls were maintained in culture with the expectation that nerve fibers from neurons of airway ganglia would remain viable, whereas fibers originating from neurons not present in the airway walls would degenerate. Airways were dissected and placed into culture dishes containing CMRL 1066 medium for 3, 5, and 7 days. In controls (noncultured), VIP‐ and SP‐like immunoreactivity was observed in nerve fibers associated with bronchial smooth muscle, glands, and blood vessel walls and in nerve cell bodies of airway ganglia. Nerve fibers containing SP were also observed within the bronchial epithelium. After 3,5, and 7 days in culture, VIP‐ and SP‐containing fibers were identified in all of the same locations except in the airway epithelium where SP‐containing fibers could not be demonstrated. VIP and SP were frequently colocalized in the same nerve fibers of bronchial smooth muscle and glands in controls and cultured airways. There were no statistically significant differences in nerve fiber density for either VIP‐ or SP‐containing fibers in bronchial smooth muscle between controlled and cultured airways. VIP concentrations in cultured airways were significantly less than in controls. The results suggest that a large proportion of VIP‐ and SP‐containing nerve fibers supplying bronchial smooth muscle, glands, and blood vessels in the airways originate from neurons of airway ganglia.