Our goal was to determine what nervous mechanisms mediate the secretion of macromolecules from tracheal submucosal glands of ferrets. To do this, we studied the secretion of 35SO4-labeled macromolecules in vitro in response to electrical or pharmacological stimulation in the absence and presence of a specific nerve blocker and autonomic antagonists. We found that electrical field stimulation and the agonists acetylcholine, phenylephrine, terbutaline, and norepinephrine each cause secretion of radiolabeled materials. The molecular weights of the labeled materials released during base line and after electrical stimulation were greater than 1,000,000. The antagonists atropine, phentolamine, and propranolol alone prevented the responses to acetylcholine, phenylephrine, and terbutaline, respectively, without preventing responses to any other of these agonists or changing baseline secretion. Only phentolamine and propranolol together prevented the response to norepinephrine. Tetrodotoxin prevented the response to electrical stimulation but not the responses to the agonists. Each of the antagonists inhibited a significant portion of the response to electrical stimulation, but the combination of all three did not completely prevent secretion. We conclude that cholinergic nerves mediate secretion via muscarinic mechanisms, that adrenergic nerves mediate secretion via both alpha- and beta-adrenergic mechanisms, and that nonadrenergic-noncholinergic nerves mediate secretion via unidentified mechanisms.