Indirect functional studies suggest that large-conductance calcium-activated potassium channels (BKCa channels) are involved in the control of ACh release from postganglionic, parasympathetic nerve terminals in the airways. The role of BKCa channels in regulating cholinergic neurotransmission was assessed by 1) investigating the effect of the putative BKCa channel opener NS1619 on cholinergic contractile responses and ACh output evoked by electrical field stimulation (EFS: 40 V, 0.5 ms, 4 Hz for 15 s every 4 min) and comparing the effect obtained with the inhibition of EFS-evoked ACh release by oxotremorine M, a muscarinic agonist, and 2) evaluating the sensitivity of these responses to the BKCa channel blocker iberiotoxin (IbTX). NS1619 (30 microM) inhibited cholinergic contractile responses by 60.0%. In contrast, NS1619 had no effect on contractile responses evoked by exogenous ACh (1 microM), which indicated that it was acting prejunctionally. NS1619 (30 microM) significantly inhibited EFS-induced ACh release by 33.9%. Oxotremorine M suppressed EFS-evoked ACh release in a concentration-dependent manner (at 1 microM, 77.4% inhibition). In neither case was the inhibition reversed by IbTX (100 nM). Collectively, the mechanical data suggest that NS1619 inhibits cholinergic contractile responses by interacting prejunctionally. The failure of IbTX to reverse the inhibitory action of NS1619 and oxotremorine M on ACh release indicates that activation of muscarinic autoinhibitory receptors is not coupled to the opening of IbTX-sensitive BKCa channels. Therefore, we propose that caution be exercised when using NS1619 as an activator of BKCa channels.