1. The guinea-pig trachea was isolated with its extrinsic innervation intact and placed in a water-jacketed dissecting dish containing warmed, oxygenated Krebs solution. The trachea was not separated from the oesophagus. Two adjacent cartilage rings of the rostral portion of the trachea were cut open opposite the trachealis and prepared for isometric tension measurements. 2. Following the addition of atropine and contraction of the trachealis with prostaglandin F2 alpha (PGF2 alpha), stimulation of the cervical sympathetic trunks elicited relaxations that were abolished by propranolol or hexamethonium. Stimulation of the vagus nerves caudal to the nodose ganglia also elicited relaxations. These vagally mediated relaxations were unaffected by propranolol but were abolished by hexamethonium or by cutting the recurrent laryngeal nerves. 3. After cutting the vagi caudal to the nodose ganglia, stimulation of the vagi rostral to the nodose ganglia elicited relaxations of the trachealis that were not significantly affected by either propranolol or hexamethonium but were abolished by cutting the superior laryngeal nerves. Stimulation of right vagi which had undergone supranodose vagotomy 14 days prior to experimentation was without effect on the smooth muscle of the guinea-pig trachea while the response to stimulation of the left vagus was unchanged. 4. Acute capsaicin desensitization abolished relaxations of the guinea-pig trachealis elicited by stimulation of the vagal fibres carried by the superior laryngeal nerves. In contrast, capsaicin desensitization only modestly inhibited relaxations elicited by stimulation of the preganglionic parasympathetic fibres carried by the recurrent laryngeal nerves and had no effect on sympathetic nerve-induced relaxations. 5. Removing the oesophagus selectively abolished relaxations elicited by stimulation of both vagal pathways of non-adrenergic relaxant innervation. Non-adrenergic relaxations of the trachealis elicited by electrical field stimulation were unaffected by removing the oesophagus. Oesophagus removal also had no effect on the parasympathetic-cholinergic contractile innervation or the sympathetic relaxant innervation of the trachealis. 6. The results indicate that the guinea-pig trachealis receives non-adrenergic relaxant innervation from both parasympathetic and capsaicin-sensitive vagal pathways. The results also suggest that the neurones mediating non-adrenergic relaxations of the trachea are sensitive to oesophagus removal. The observation that oesophagus removal abolishes parasympathetic relaxations of the trachealis while having no effect on parasympathetic contractions supports the hypothesis that the guinea-pig trachealis receives excitatory and inhibitory innervation from distinct vagal parasympathetic pathways.