The contractile effects of substance P (SP) were studied in isolated rabbit tracheal smooth muscle (TSM) segments in vitro. Noncumulative administration of SP produced dose-dependent increases in TSM tension. The mean (+/- SE) peak isometric tension (Tmax) with SP was 35.7 (+/- 6.2%) of the corresponding Tmax response to methacholine. The dose of agonist producing 50% of Tmax (ED50) was significantly lower for SP, averaging 1.8 (+/- 0.4) X 10(-7) M, vs. 1.7 (+/- 0.32) X 10(-6) M for methacholine. Blockade of both parasympathetic ganglia with hexamethonium (10(-4) M) and neural transmission with tetrodotoxin (1 microgram/ml) had no effect on the TSM response to SP. On the other hand, TSM contraction to an ED50 dose of SP was 1) augmented by a mean (+/- SE) of 470 (+/- 110%) following pretreatment with the cholinesterase inhibitor, neostigmine (10(-6) M);2) inhibited by a mean (+/- SE) of 35 (+/- 15%) with the cholinergic antagonist, atropine (10(-4) M); and 3) also inhibited by a mean (+/- SE) of 45 (+/- 11%) following inhibition of acetylcholine synthesis with hemicholinium-3 (10(-4) M). Antagonists to 5-hydroxytryptamine, alpha 1-adrenergic, and histamine receptor binding had no effect on TSM contraction with SP. In contrast, the SP antagonist, D-Pro2,D-Trp7,9-SP, markedly inhibited TSM contraction to SP. Our findings indicate that rabbit TSM is sensitive to SP and its contraction is in part mediated by a peripheral cholinergic action, likely involving the accelerated release of acetylcholine at the airway neuromuscular junction.