Purinergic stimulation of airway epithelial cells induces Cl- secretion and modulates Na+ absorption by an unknown mechanism. To gain insight into this mechanism, we used a perfused micro-Ussing chamber to assess transepithelial voltage (V(te)) and amiloride-sensitive short-circuit current (I(sc-Amil)) in mouse trachea. Exposure to apical ATP or UTP (each 100 micromol/l) caused a large initial increase in lumen negative V(te) and I(sc), corresponding to a transient Cl- secretion, while basolateral application of ATP/UTP induced only a small secretory response. Luminal, but not basolateral, application of nucleotides was followed by a sustained and reversible inhibition of I(sc-Amil) that was independent of extracellular Ca2+ or activation of protein kinase C and was not induced by carbachol (100 micromol/l) or the Ca2+ ionophore ionomycin (1 micromol/l). Removal of extracellular Cl- or exposure to 200 microM DIDS reduced UTP-mediated inhibition of I(sc-Amil) substantially. The phospholipase inhibitor U73122 (10 micromol/l) and pertussis toxin (PTX; 200 ng/ml) both attenuated UTP-induced Cl- secretion and inhibition of I(sc-Amil). Taken together, these data imply a contribution of Cl- conductance and PTX-sensitive G proteins to nucleotide-dependent inhibition of the amiloride-sensitive Na+ current in the mouse trachea.