We developed a pleural surface fluorescence method to measure Na⁺ and Cl⁻transport in perfused mouse lungs. The air space was filled with aqueous fluid containing membrane-impermeant fluorescent indicators of Cl⁻ (lucigenin) or Na⁺ (Sodium Green). After instillation of a Cl⁻-free solution into the air space, an increase in perfusate Cl⁻ concentration from 0 to 30 mM produced a decrease in surface lucigenin fluorescence (6.5%/min) corresponding to Cl⁻ influx of 1.0 mM/min. Cl⁻ influx was increased to 2.1 ± 0.3 mM/min by forskolin, and the increase was inhibited by glibenclamide. cAMP-stimulated Cl⁻ influx was decreased by 57% in CFTR null mice. After instillation of a Na⁺-free solution into the air space, an increase in perfusate Na⁺ concentration from 0 to 30 mM gave increased Sodium Green fluorescence (Na⁺ influx of 1.2 mM/min), which increased approximately fivefold after cAMP agonists. Cl⁻ and Na⁺transport were not affected in lungs from mice lacking aquaporins AQP1 or AQP5. Our results establish a pleural surface fluorescence method to measure unidirectional Cl⁻ and Na⁺ flux in intact lung and provide evidence for cAMP-stimulated transcellular Cl⁻ and Na⁺ transport.