The present study was conducted to determine if acetylcholine (ACh) release from airway cholinergic nerves is increased and if modulation of ACh release by prejunctional receptors is altered in horses with heaves, an obstructive airway disease characterized by airway inflammation and bronchospasm. Trachealis strips and bronchial segments of normal horses and horses affected with heaves were suspended in 2-ml tissue baths. ACh release was induced by electrical field stimulation and the bath ACh content was measured by high performance liquid chromatography (HPLC) with electrochemical detection. In response to electrical field stimulation, the rate of ACh release from cholinergic nerves innervating the trachea was not significantly different between the two groups of horses. The nonselective muscarinic receptor antagonist atropine augmented ACh release to the same extent in both groups, indicating that there is no dysfunction of muscarinic autoreceptors on cholinergic nerves of large airways from horses with heaves. Compared with the data from normal horses, the inhibitory effect of the alpha 2-adrenoceptor agonist clonidine on ACh release was lacking in the bronchi and less potent in the trachea, suggesting that the prejunctional alpha 2-adrenoceptors are dysfunctional in horses with heaves. Neither exogenous prostaglandin E2 (PGE2) nor the cyclooxygenase inhibitor indomethacin influenced ACh release from the bronchi, suggesting that a decrease in airway mucosal PGE2 production reported previously in horses with heaves does not alter ACh release.