1. Muscarinic receptor subtypes mediating neurogenic mucus secretion in ferret trachea were characterized in vitro and in vivo using 35SO4 as a label for secreted mucus, and the muscarinic receptor antagonists telenzepine for the M1 receptor subtype, methoctramine for the M2 subtype and 4-diphenylacetoxy-N-methylpiperidine methobromide (4-DAMP) for the M3 receptor. We also performed receptor binding and mapping studies. 2. Each muscarinic antagonist displaced [N-methyl-3H]scopolamine binding with high-affinity binding constant (KH) values of 1.9, 2.7 and 5.0 nM for telenzepine, methoctramine and 4-DAMP, respectively. Muscarinic M1 and M3 receptors localized to submucosal glands, whereas M2 receptors did not. 3. In vitro, electrical stimulation (50 V, 10 Hz, 0.5 ms for 5 min) increased 35SO4 output by 160%. Telenzepine did not inhibit the neurogenic secretory response at concentrations two-or twentyfold its KH value, nor did it inhibit secretion induced by acetylcholine (ACh). 4-DAMP inhibited neurogenic secretion by 80 and 95%, respectively, at concentrations two-and twentyfold its KH value, and also inhibited ACh-induced secretion. Methoctramine potentiated neurogenic secretion induced at 2.5 Hz (50 V, 0.5 ms for 5 min) in a dose-related (5.4-100 nM) manner with increases of 33-451% above electrically stimulated values. Methoctramine did not potentiate secretion induced at 10 Hz and did not have any effect on ACh-induced secretion. 4. In vivo, vagal stimulation (10 V, 10 Hz, 2 ms for 8 min) increased output of 35SO4 by approximately 120%. Telenzepine had no significant effect on neurogenic secretion. Methoctramine approximately doubled the stimulated response, whereas 4-DAMP abolished the stimulated secretory response. 5. We conclude that in ferret trachea, cholinergic nerve stimulation increases mucus secretion via muscarinic M3 receptors on the submucosal glands. The magnitude of the secretory response is regulated by neuronal M2 muscarinic receptors. The muscarinic M1 receptors localized to the submucosal glands do not appear to be involved with mucus secretion.