CFTR is the major Cl- channel in several epithelia, but its importance in airway cells and its interactions with other channels are still poorly understood. We studied the role of CFTR and other Cl- channels in two well-differentiated human airway cell lines--16HBE and Calu-3. Both lines form tight junctions, and the Calu-3 line exhibits large currents in the Ussing chamber that represent Cl- secretion. Apical membrane Cl- conductance in both cell lines in mediated primarily and possibly exclusively by CFTR, because (1) CFTR channels are almost the only anion channels observed in cell-attached patches from confluent cells, (2) whole-cell Cl- current has a linear, time-independent signature, and (3) blockers of many Cl- channels, such as DIDS, do not block Isc. Outwardly rectifying (ORDIC) channels were identified in both airway cell lines. Activation of ORDIC channels in Calu-3 cells required 3 conditions: (1) stimulation with isoproterenol, (2) excision and depolarization, and (3) prior treatment of the cell to disrupt tight junctions. To further assess the physiological significance of ORDIC channels, we compared cell lines which have > 10-fold difference in functional expression of ORDIC channels. ORDIC channel expression was not associated with swelling-activated Cl- currents.