The neuromodulatory actions of Septide, a selective neurokinin-1 (NK-1) agonist, and substance P (SP) were investigated in isolated rabbit tracheal smooth muscle (TSM) segments. The tissues were placed in organ baths containing modified Krebs-Ringer solution and contracted with either the agonists or electrical field stimulation (ES) with frequencies ranging from 1 to 50 Hz. The neutral endopeptidase (NEP) inhibitor, phosphoramidon (10(-6) M), had no significant effect on Septide-mediated contractions. Septide-mediated contractions were augmented in the presence of neostigmine, eliminated in the presence of atropine, and diminished in the presence of tetrodotoxin. Both SP and Septide increased ES-induced contractions in a dose-dependent manner. On the other hand, the presence of both Septide and SP did not further augment this neuromodulatory action. After NK-1 desensitization, Septide-mediated contractions were also virtually eliminated; however, the peptide's neuromodulatory action was unaffected. In contrast, the presence of GR-82334, a selective NK-1 antagonist, eliminated Septide's neuromodulatory activity. These findings provide evidence that 1) NK-1 receptors facilitate both the direct release of ACh as well as augment the release of ACh by ES and 2) the cholinergic pathways involved with these two processes may represent different mechanisms.