Effects of nonadrenergic and noncholinergic (NANC) inhibitory nerves on cholinergic neurotransmission were examined in isolated bronchial segments from cats in the presence of propranolol (10(-6) M) and indomethacin (10(-6) M) by use of electrical field stimulation (EFS) techniques. EFS caused contraction alone in tissues at the baseline tension and biphasic responses (contraction and relaxation) in tissues precontracted with 5-hydroxytryptamine. Contraction was abolished by atropine (10(-6) M), and relaxation was abolished by tetrodotoxin (10(-6) M). At the baseline tension, EFS at frequencies greater than 10 Hz inhibited the subsequent (4 min later) contraction induced by EFS at 1-5 Hz. EFS-induced inhibition was stimulus frequency dependent and reached maximum at 20 Hz. However, EFS at 20 Hz did not inhibit the subsequent contractile response to acetylcholine (10(-7) to 10(-3) M). Exogenously applied vasoactive intestinal peptide mimicked EFS-induced inhibitory effects, but substance P and calcitonin gene-related peptide did not. The inhibitory effect of EFS at 20 Hz was not altered by pyrilamine, cimetidine, naloxone, methysergide, phentolamine, BW755C, AF-DX 116, or removal of epithelium. These results imply that the NANC transmitter acts via presynaptic cholinergic receptors.