We investigated the signal transmission pathway by which activation of μ-opioid receptors attenuates acetylcholine (ACh) release in bovine trachealis. Electrical stimulation (ES)-induced [3H]-ACh release was determined in bovine tracheal smooth muscle strips pre-incubated with either the Gi-protein inhibitor pertussis toxin (PTX, 500ng/ml and 1μg/ml) or the Gz-protein specific inhibitor arachidonic acid (AA, 10−6M and 10−5M) and then treated with DAMGO (D-Ala2,N-MePhe4,Gly-ol5-enkephalin) 10−5M. Indomethacin 10−5M was used to block AA cascade. The inhibitory effect of DAMGO on ES-induced [3H]-ACh release was PTX-insensitive, but, by contrast, ablated by AA in a concentration-dependent manner. AA 10−5M alone reduced [3H]-ACh release, an effect that was prevented by iberiotoxin 10−7M, suggesting an involvement of Ca2+-activated K+-channels. Western blot analysis consistently showed immunoreactive bands against a specific antibody anti-Gz-α subunit at ∼40kDa, consistent with the presence of Gz-protein. The present findings suggest that in isolated bovine trachealis, activation of μ-opioid receptors inhibits ACh-release through a signal transmission pathway involving Gz-protein rather than Gi-protein.