To clarify the mechanism for Cl- transport in swine tracheal submucosal gland cells, we measured chloride (Cl-) flux by means of a Cl--sensitive fluorophore, 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ). An abrupt change of Cl--free bath to a Cl--containing (114 mM) solution induced the Cl- influx into the cells at a rate of 0.52 mM/s (p < 0.05). Removal of sodium (Na+) and the addition of 0.5 mM furosemide significantly reduced the rate of gradient-induced Cl- influx to 0.21 (p < 0.05) and 0.19 mM/s (p < 0.05), respectively. In gland tissue treated with 0.1 mM ouabain, the removal of external potassium (K+) in the presence of 5 mM barium significantly reduced the rate of Cl- influx to 0.21 mM/s (p < 0.05). An abrupt change from a Cl--containing bath to a Cl--free solution induced the Cl- efflux from the cells at a rate of 0.10 mM/s (p < 0.05). Acetylcholine increased the rate of gradient-induced Cl- efflux in a dose-dependent manner to 0.78 mM/s at 10-6M This effect of acetylcholine was significantly diminished by diphenylamine-2-carboxylic acid (DPC, 10-9M), a Cl- channel blocker (p < 0.05). Isoproterenol (10-5 M) had no effect on the Cl- efflux. These findings indicate that a Na-K-Cl co-transporter plays a major role in Cl- entry, and that Cl- efflux was activated by cholinergic receptor stimulation, but not by β-adrenergic stimulation, via a DPC-inhibitable Cl- channel