1. Deletion of phenylalanine at position 508 (DeltaF508) of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is the most common mutation causing cystic fibrosis (CF). Effective pharmacological therapy of CF caused by the DeltaF508-CFTR mutation requires the rescue of both intracellular processing and channel gating defects. 2. We identified a class of natural coumarin compounds that can correct the defective DeltaF508-CFTR chloride channel gating by screening a collection of 386 single natural compounds from Chinese medicinal herbs. Screening was performed with an iodide influx assay in Fischer rat thyroid epithelial cells coexpressing DeltaF508-CFTR and an iodide-sensitive fluorescent indicator (YFP-H148Q/I152L). 3. Dose-dependent potentiation of defective DeltaF508-CFTR chloride channel gating by five coumarin compounds was demonstrated by the fluorescent iodide influx assay and confirmed by an Ussing chamber short-circuit current assay. Activation was fully abolished by the specific CFTR inhibitor CFTR(inh)-172. Two potent compounds, namely imperatorin and osthole, have activation K(d) values of approximately 10 micromol/L, as determined by the short-circuit current assay. The active coumarin compounds do not elevate intracellular cAMP levels. Activation of DeltaF508-CFTR by the coumarin compounds requires cAMP agonist, suggesting direct interaction with the mutant CFTR molecule. Kinetics analysis indicated rapid activation of DeltaF508-CFTR by the coumarin compounds, with half-maximal activation of < 5 min. The activating effect was fully reversed for all five active compounds 45 min after washout. 4. In conclusion, the natural coumarin DeltaF508-CFTR activators may represent a new class of natural lead compounds for the development of pharmacological therapies for CF caused by the DeltaF508 mutation.