To investigate the effects of changes in intracellular cAMP on ₂-adrenoceptor (AR)-induced inhibition of airway acetylcholine (ACh) release, we examined the effects of the ₂-AR agonist clonidine on electrical field stimulation-evoked ACh release from equine tracheal parasympathetic nerves before and after treatment with 8-bromo-cAMP or forskolin. We also tested whether charybdotoxin (ChTX)- or iberiotoxin (IBTX)-sensitive Ca²⁺-activated K⁺ channels mediate ₂-AR-induced inhibition by examining the effect of clonidine in the absence and presence of ChTX or IBTX on ACh release. The amount of released ACh was measured by HPLC coupled with electrochemical detection. Clonidine (10⁷ to 10⁵ M) dose dependently inhibited ACh release before and after treatment with 8-bromo-cAMP (10³ M) or forskolin (3 × 10⁵ M). ChTX and IBTX, both at the concentration of 5 × 10⁷ M, significantly increased ACh release; however, they did not alter the magnitude of clonidine-induced inhibition. These results indicated that in equine tracheal parasympathetic nerves, ₂-AR-induced inhibition of ACh release is via an intracellular cAMP-independent pathway. Activation of both ChTX- and IBTX-sensitive Ca²⁺-activated K⁺ channels inhibits the electrical field stimulation-evoked ACh release, but these channels are not involved in the ₂-AR-induced inhibition of ACh release.