The cystic fibrosis (CF) gene codes for CF transmembrane regulator (CFTR), a small-conductance linear Cl- channel, but numerous studies have identified a larger conductance, rectifying Cl- channel as the adenosine 3',5'-cyclic monophosphate (cAMP)-regulated channel that is defective in airway cells. We examined Cl- conductance in a bronchial epithelial cell line that expresses CFTR, 16HBE14o-, (CFTR+) and in an airway cell line that does not, 9HTEo-/S, (CFTR-). Ionomycin or hypotonic Ringer increased iodide efflux from both cell lines; however, forskolin increased iodide efflux or whole cell Cl- currents only in CFTR+ cells. Forskolin-stimulated whole cell currents were linear, voltage independent, and blocked by iodide. Cell-attached and outside-out patches from confluent CFTR+ but not CFTR- cells revealed 6-pS channels having linear current-voltage relations, permselectivity Cl > I (partial block by external iodide), and little or no inhibition by 5-nitro-2-(3-phenylpropylamino)-benzoate. The number of active channels per patch increased from 0.6 to 3.0 after forskolin. Channels closed after excision with tau = 4 s, but activity could be prolonged with ATP or protein kinase A plus ATP. Channels were modeled with one open and four closed states and show apparent cooperativity in gating. Rectifying Cl- channels previously implicated in CF were not seen in cell-attached recordings from either cell line but were abundant in excised patches from both cell lines. Thus CFTR channels are the pathway for cAMP-mediated Cl- conductance in these human airway cells, Ca2+ and swelling-induced channels do not require CFTR, and CFTR-cells display a CF phenotype.