The present study was undertaken to investigate the functional presence of inhibitory muscarinic M2 autoreceptors on postganglionic cholinergic nerve endings in different generations of human airways. To this end, the effects of the M2-selective muscarinic receptor antagonists AQ-RA 741 and gallamine were studied on electrical field stimulation-induced twitch contractions of preparations from trachea and from bronchial airways of varying diameter. Furthermore, electrically evoked release of endogenous acetylcholine from human bronchial preparations, and the effect of the muscarinic receptor antagonist atropine thereon, was measured by high-performance liquid chromatography. On average, twitch contractions were significantly but only slightly (11 to 15%) potentiated by M2-selective concentrations of AQ-RA 741 and gallamine, despite approximately half of the preparations showing no potentiation at all. A subdivision into airway generations showed that M2 autoreceptor function was not readily detectable in bronchioles and subsegmental bronchi. By contrast, both with AQ-RA 741 and gallamine a clear potentiation (26 to 36%) of the twitch contractions was observed in approximately half of the terminal bronchi and in all central airway preparations. Moreover, the evoked release of endogenous acetylcholine in terminal and subsegmental bronchi was significantly facilitated by atropine, to 162 to 189% of controls. These results provide strong and partly direct evidence for the existence of inhibitory muscarinic M2 receptors on postganglionic cholinergic nerve endings in human central airways and subsegmental and terminal bronchi, but not in bronchioli. It remains to be established, however, why these M2 receptors exhibit a rather variable functionality in regulating cholinergic nerve-mediated contraction in different airway generations.