To determine whether serous or mucous cells in tracheal submucosal glands respond to the neuropeptides substance P (SP) and vasoactive intestinal peptide (VIP), we studied the peptide-induced changes in gland cell morphology accompanying release of 35SO4-labeled macromolecules from tracheal explants of ferrets. Explants were labeled for 1 h in medium containing 35SO4 and washed for 3.5 additional hours. Base-line secretion in the absence of drugs declined between 1.5 and 3.5 h after the pulse. Between 2.5 and 3.5 h, the average percent change in counts per minute recovered per sample period was not significantly different from zero (P greater than 0.3; n = 6). Substance P (10(-5) M) and VIP (2 X 10(-6) M) added 4 h after labeling each increased greatly the release of 35SO4-labeled macromolecules (SP, 219%; VIP, 180%) above base line. Bethanechol, a muscarinic-cholinergic agonist (10(-5) M), increased secretion by an average of 142% above base line (each effect, P less than 0.05; n = 6 each). Light and electron microscopy of the control tissues showed glands with narrow lumens and numerous secretory granules. Glands treated with SP or VIP had enlarged lumens and the serous cells were markedly degranulated. These phenomena were documented by morphometry and suggest that SP and VIP cause secretion from glands at least partially by stimulating exocytosis from serous cells.