To investigate presynaptic, regulatory mechanisms on parasympathetic nerve fibres innervating the airways, the release of newly-synthesized [3H]acetylcholine from the isolated trachea was studied. Reverse phase HPLC followed by liquid scintillation spectrometry was used to separate and quantify the radioactive compounds choline, phosphorylcholine and acetylcholine in the incubation medium and the tissue. During the incubation of the tracheae with [3H]choline a significant synthesis of [3H]acetylcholine (35,000 dpm/preparation) and [3H]phosphorylcholine (500,000 dpm/preparation) occurred. In epithelium-deficient tracheae the formation of [3H]phosphorylcholine was enhanced, whereas the content of [3H]acetylcholine remained unchanged. The spontaneous outflow of tritium consisted mainly of [3H]phosphorylcholine (900 dpm/3 min) and [3H]choline (800 dpm/3 min); [3H]acetylcholine was only a minor fraction (50 dpm/3 min). Electrical stimulation of tracheae with intact epithelium caused only a small release of [3H]acetylcholine (460 dpm in the sample obtained during stimulation), but a considerable outflow of [3H]phosphorylcholine (1,900 dpm) without affecting the outflow of [3H]choline. Electrical stimulation of epithelium-deficient tracheae, however, induced a substantial release of [3H]acetylcholine (2,400 dpm), but only a small outflow of [3H]phosphorylcholine. Chemical stimulation (30 mumol/l veratridine) also caused a large release of [3H]acetylcholine (1,700 dpm) without affecting the outflow of [3H]phosphorylcholine or [3H]choline. Indomethacin (3 mumol/l) enhanced the electrically-evoked release of [3H]acetylcholine from tracheae with intact epithelium by 89%. The present experiments demonstrate a strong inhibition by the epithelium of the electrically-evoked release of [3H]acetylcholine from the isolated guinea-pig trachea. Cyclooxygenase products of arachidonic acid do not appear as the main mediators of the epithelium-derived inhibition of acetylcholine release.