The dominant route for Cl secretion in mouse tracheal epithelium is via Cl channels different from the cystic fibrosis (CF) transmembrane conductance regulator (CFTR), the channel that is defective in CF. It has been proposed that the use of purinergic agonists to activate these alternative channels in human airways may be beneficial in CF. In the present study, two conditionally immortal epithelial cell lines were established from the tracheae of mice possessing the tsA58 T antigen gene, one of which [MTE18-(/)] was homozygous for a knockout of CFTR and the other [MTE7b-(+/)] heterozygous for CFTR expression. In Ussing chamber studies, amiloride (10⁴ M) and a cocktail of cAMP-activating agents (forskolin, IBMX, and dibutyryl cAMP) resulted in small changes in the short-circuit current (I_sc) and resistance of both cell lines, with larger increases in I_sc being elicited by ionomycin (10⁶ M). Both cell lines expressed P₂Y₂ receptors and responded to the purinergic agonists ATP, UTP, and 5'-adenylylimidodiphosphate (10⁴ M) with an increase in I_sc. This response could be inhibited by DIDS and was abolished in the presence of Cl-free Ringer solution. Reducing the mucosal Cl concentration increased the response to UTP of both cell lines, with a significantly greater increase in MTE18-(/) cells. Pretreatment of these cells with thapsigargin caused a direct increase in I_sc and inhibited the response to UTP. These data suggest that both cell lines express purinergic-regulated Cl currents and may prove valuable tools in studying the properties of this pathway.