1. Membrane currents activated by acetylcholine (ACh) were investigated in isolated canine and guinea-pig tracheal myocytes using the nystatin perforated patch configuration of whole-cell recording. ACh caused depolarization accompanied by a membrane conductance increase. 2. When cells were held under voltage clamp (holding potential, Vh = -60 mV), ACh elicited inward current (IACh) of up to 3900 pA, with a reversal potential (Erev) of approximately -20 mV. 3. Removal of extracellular Na+ (Na+o) reduced but did not eliminate IACh. IACh remaining in the absence of Na+ reversed direction close to the predicted equilibrium potential for Cl-. Erev shifted 32 +/- 4 mV per 10-fold change of [Cl-]i. Increasing external [K+] caused Erev to shift in the positive direction. These results suggest that ACh activated chloride and non-selective cation conductances. 4. In the absence of Na+o, the Cl- channel blockers SITS or niflumic acid reversibly antagonized IACh. 5. Caffeine and ryanodine elicited currents both in the presence and absence of Na+o; these currents had a reversal potential similar to that of IACh. Caffeine applied before ACh occluded the response to ACh. 6. We also observed two types of spontaneous membrane currents. Spontaneous transient outward currents (STOCs) may represent Ca(2+)-activated K+ currents. Spontaneous inward currents were also observed which were reduced in magnitude (but not eliminated) by removal of Na+o and reversed direction at approximately the Cl- equilibrium potential. The spontaneous inward currents and STOCs were coincident and were reversibly suppressed by ACh. 7. ACh elicited contractions of cells under voltage clamp at -60 mV, an effect also observed in the absence of extracellular Ca2+ or when IACh was reduced by omission of Na+o and exposure to Cl- channel blockers. The number of cells which did contract in response to ACh decreased, however, when the concentration of internal Cl- decreased. 8. All effects of ACh on contraction and membrane currents were antagonized by atropine. 9. We conclude that activation of muscarinic receptors in mammalian tracheal myocytes causes release of Ca2+ from intracellular stores and subsequent activation of Cl- and non-selective cation conductances. This is the first direct demonstration of these conductances in tracheal smooth muscle cells. Activation of these conductances does not appear to be required for contraction. However, regulation of cytosolic Cl- levels may be important for release and uptake of Ca2+ from internal stores.