Cultured ovine trachea submucosal gland cells release lysozyme in response to extracellular application of secretagogues, including the muscarinic receptor agonist methacholine (20 microM). Investigation of the ultrastructure has shown that these cells contain electron‐dense cored granules, which differ from the intact tissue, but appear to be released in response to the application of methacholine and can be arrested during exocytosis by the application of tannic acid. The release process appears to be linked to electrophysiological events activated by methacholine. Extracellular application of methacholine and intracellular photorelease of Ca2+ from DM‐nitrophen evoked similar events suggesting that a rise in intracellular Ca2+ may occur following muscarinic receptor activation. Measurements of the reversal potential and the inhibitory action of the chloride channel blocker niflumic acid (10 microM) indicated that Ca(2+)‐activated Cl‐ channel activity underlies these events. Some of the cultured submucosal gland cells also responded similarly to intracellular photorelease of inositol 1,4,5‐trisphosphate, suggesting a possible link between muscarinic receptor occupation by agonist, release of calcium from stores, and activation of Ca(2+)‐activated Cl‐ current. Secretion of lysozyme, methacholine‐activated currents and currents evoked by intracellular photorelease of Ca2+ were also attenuated by the potent bronchodilator Ro 31‐6930 (1 microM). We conclude that Ca(2+)‐activated Cl‐ conductances play an important role in secretory processes in cultured submucosal gland cells. This may have a bearing on both physiological control of secretory events and regulation of the nature of airway surface liquid. Ca(2+)‐activated Cl‐ channels may offer a potential target site for novel therapeutic agents.