1. Several agonists modulate cholinergic neurotransmission in airways raising the possibility that there may be a common inhibitory mechanism, such as the activation of a common K+ channel in the nerve ending. To test this hypothesis, we examined whether blockers of K+ channels are able to depress the prejunctional inhibitory modulation of cholinergic contractile responses by various agonists in guinea-pig and human airways in vitro. 2. Electrical field stimulation (40 V, 0.5 ms) was applied to guinea-pig (0.5 Hz) or human (1 Hz) tracheal strips every 4 min to elicit cholinergic neural responses. The effects of the K+ channel blockers, charybdotoxin (ChTX, 10 nM), apamin (100 nM) and glibenclamide (1 microM), on the prejunctional inhibition of cholinergic contraction evoked by neuropeptide Y (NPY, 100 nM), an alpha 2-agonist, clonidine (10 nM), a mu-opioid agonist, [D-Ala2, NMePhe4, Gly-ol5]-enkephalin (DAMGO, 100 nM), and a KATP channel opener, lemakalim (300 nM) were tested in guinea-pigs. In human tissues, the effect of ChTX (10 nM) on the mu-opioid (DAMGO, 300 nM)-induced inhibition of cholinergic nerves was examined. 3. In guinea-pigs, ChTX (10 nM) significantly reversed the prejunctional inhibition of cholinergic contraction by NPY (84.2 +/- 16.2%), clonidine (71.9 +/- 22.4%), DAMGO (67.3 +/- 13.1%) and lemakalim (20.9 +/- 9.4%) (n = 5, P < 0.05, respectively), while apamin (100 nM) had no effect. Glibenclamide (10 microM) reduced only the lemakalim-induced inhibitory modulation. ChTX (10 nM) itself potentiated cholinergic contraction (24.6 +/- 9.4%, n = 5, P < 0.05) without affecting exogenously applied acetylcholine dose-response curves. Pretreatment with ChTX (10 nM) significantly reduced the inhibitory modulation of cholinergic nerves by NPY, clonidine and DAMGO, but not by lemakalim. 4. In human tissues, ChTX significantly reduced DAMGO-induced prejunctional inhibition of cholinergic contraction (13.6 +/- 8.5% with and 46.5 +/- 5.5% without ChTX, respectively; n = 5, P < 0.05). 5. These results may support a hypothesis that the activation of ChTX-sensitive K+ channels is involved in the inhibitory modulation of cholinergic neuro-transmission by agonists acting on presynaptic receptors in guinea-pig and human airways.