The role of chloride channels has been examined in canine tracheal smooth muscle by recording mechanical responses to field stimulation and to acetylcholine (ACh) and by sucrose gap recording of excitatory junction potentials and ACh-induced electrical changes. The results of substitution studies using isethionate for chloride provided evidence that a chloride conductance contributes to the resting potential. The extrapolated reversal potential for ACh-induced depolarization was positive to the resting potential. Isethionate substitution inhibited ACh-induced depolarization, consistent with a contribution from increased Cl- conductance to the depolarization induced by ACh. However, closure of K+ channels and opening of a non-specific cation channel could also contribute to depolarization. Further study of the effects of isethionate substitution during prolonged tissue exposure to chloride-free medium showed that retention or the accumulation of Ca2+ in intracellular stores was impaired. We conclude that effects of chloride deprivation on responses to ACh may reflect an early increase in Cl- conductance, but longer term changes reflect the requirement for this anion to maintain internal Ca2+ stores.