We investigated the regulation of the large-conductance Ca(2+)-dependent K+ (KCa) channel by acetylcholine (ACh) in freshly dissociated tracheal smooth muscle cells. Channels were recorded in the cell-attached patch configuration, and cells were stimulated with ACh, muscarine, or caffeine. We identified KCa channel activity based on 1) the voltage dependence of channel opening; 2) the large unitary conductance (242 +/- 5 pS with symmetrical 135 mM K+); 3) dependence of the reversal potential on the [K+] gradient, shifting 56 +/- 3 mV/10-fold change in extracellular [K+]; and 4) opening of channels after elevation of cytosolic free Ca2+ concentration ([Ca2+]i) using the Ca2+ ionophore A23187. When cells were bathed either in a physiological saline solution or a solution containing 135 mM K+ (to clamp cell membrane potential near 0 mV), ACh caused contraction of cells and activation of voltage-dependent channels. With 135 mM extracellular K+, the channels activated by ACh had a unitary conductance of 247 +/- 10 pS, and currents reversed near the K+ equilibrium potential (-1 +/- 1 mV). The effects of ACh were reversible, blocked by atropine, and mimicked by muscarine. From these characteristics we conclude that muscarinic stimulation of canine tracheal smooth muscle cells leads to activation of the large-conductance KCa channel. Because the KCa channels were isolated from ACh by the patch pipette, the increased channel activity was probably mediated by a cytosolic second messenger. ACh shifted the threshold for KCa channel opening to less positive membrane potentials, similar to that seen with elevation of [Ca2+]i.(ABSTRACT TRUNCATED AT 250 WORDS)