1. Neuropeptide Y (NPY) is localized to adrenergic nerves in guinea-pig airways but its function is not known. 2. NPY (1 X 10(-10)-3 X 10(-7) M) had no direct effect on guinea-pig tracheal smooth muscle in vitro. 3. NPY produced a concentration- and frequency-dependent inhibition of the cholinergic component of responses elicited by electrical field stimulation (EFS) whilst having no effect on the contractile response to exogenously applied acetylcholine (ACh). 4. Yohimbine was able to reverse significantly the inhibitory effect of noradrenaline on the cholinergic component to EFS without having any significant effect on the inhibition produced by NPY. 5. Neither blockade of beta-adrenoceptors by propranolol, nor depletion of adrenergic nerves by incubation with 6-hydroxydopamine caused any significant alteration in the response to EFS in the presence of 3 X 10(-7) M NPY. Bretylium tosylate incubation to prevent noradrenaline release produced a small but significant enhancement of the inhibitory effect of NPY on EFS at high frequencies. 6. NPY appears to reduce the cholinergic component to EFS via a prejunctional mechanism, acting directly on receptors on cholinergic nerve terminals, rather than affecting adrenergic mechanisms. NPY released by adrenergic nerves may modulate cholinergic neurotransmission in guinea-pig airways.