1. Hen tracheal epithelium can be stimulated by serosal application of acetylcholine (ACh) to secrete Cl- equal to approximately 60-90 microA/cm2. 2. Radio-ligand-displacement for IP3, cAMP and cGMP and ion channel selective drugs in voltage clamp set-ups were employed to characterize second messengers and Cl-, K+ and Ca2+ channels involved in the ACh response. 3. ACh induced a significant rise in IP3 in isolated tracheocytes, while ACh did not influence the production of cAMP in whole tissue, isolated tracheocytes or basolateral cell membrane vesicles. Further ACh desensitization did not effect cAMP level in tracheocytes. In addition neither ACh stimulation nor desensitization interfered with cAMP production in presence of 4.5 microM forskolin in tracheocytes, a level of forskolin rising base level cAMP by around five fold. 4. Around 35% of ACh Cl- secretion depends on Ca2+ mobilization from internal stores and about 65% on Ca2+ influx over basolateral membrane. The activated Ca2+ channel is insensitive to class I, II, III and IV Ca2+ antagonists. 5. A 23187 can mimic the ACh effect although 30% is indomethacin-sensitive demonstrating a prostaglandin activated adenylyl cyclase. 6. Two K+ channels are involved in ACh secretion, one sensitive to Ba2+ and quinine and both insensitive to 4-aminopyridine, apamin, charybdotoxin and TEA. 7. Flufenamate and triaminopyrimidine block a non-selective ion channel likely involved in the ACh response. An ACh activated apical Cl- channel is NPPB-sensitive.