The active absorption of fluid from the airspaces of the lung is important for the resolution of clinical pulmonary edema. Although ENaC channels provide a major route for Na⁺ absorption, the route of Cl⁻ transport has been unclear. We applied a series of complementary approaches to define the role of Cl⁻ transport in fluid clearance in the distal airspaces of the intact mouse lung, using wild-type and cystic fibrosis ΔF508 mice. Initial studies in wild-type mice showed marked inhibition of fluid clearance by Cl⁻ channel inhibitors and Cl⁻ ion substitution, providing evidence for a transcellular route for Cl⁻ transport. In response to cAMP stimulation by isoproterenol, clearance was inhibited by the CFTR inhibitor glibenclamide in both wild-type mice and the normal human lung. Although isoproterenol markedly increased fluid absorption in wild-type mice, there was no effect in ΔF508 mice. Radioisotopic clearance studies done at 23°C (to block active fluid absorption) showed ∼20% clearance of ²²Na in 30 min both without and with isoproterenol. However, the clearance of ³⁶Cl was increased by 47% by isoproterenol in wild-type mice but was not changed in ΔF508 mice, providing independent evidence for involvement of CFTR in cAMP-stimulated Cl⁻ transport. Further, CFTR played a major role in fluid clearance in a mouse model of acute volume-overload pulmonary edema. After infusion of saline (40% body weight), the lung wet-to-dry weight ratio increased by 28% in wild-type versus 64% in ΔF508 mice. These results provide direct evidence for a functionally important role for CFTR in the distal airspaces of the lung.