Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) protein that reduce cAMP-stimulated Cl(-) conductance in airway and other epithelia. The purpose of this investigation was to identify new classes of potent CFTR activators. A collection of 60,000 diverse drug-like compounds was screened at 10 microm together with a low concentration of forskolin (0.5 microm) in Fisher rat thyroid epithelial cells co-expressing human CFTR and a green fluorescent protein-based Cl(-) sensor. Primary screening yielded 57 strong activators (greater activity than reference compound apigenin), most of which were unrelated in chemical structure to known CFTR activators, and 284 weaker activators. Secondary analysis of the strong activators included analysis of CFTR specificity, forskolin requirement, transepithelial short-circuit current, activation kinetics, dose response, toxicity, and activation mechanism. Three compounds, the most potent being a dihydroisoquinoline, activated CFTR by elevating cellular cAMP, probably by phosphodiesterase inhibition. Fourteen compounds activated CFTR without cAMP elevation or phosphatase inhibition, suggesting direct CFTR interaction. The most potent compounds had tetrahydrocarbazol, hydroxycoumarin, and thiazolidine core structures. These compounds induced CFTR Cl(-) currents rapidly (<5 min) with K(d) down to 200 nm and were CFTR-selective, reversible, and nontoxic. Several compounds, the most potent being a trifluoromethylphenylbenzamine, activated the CF-causing mutant G551D, but with much weaker affinity (K(d) > 10 microm). When added for 10 min, none of the compounds activated DeltaPhe(508)-CFTR in transfected cells grown at 37 degrees C (with DeltaPhe(508)-CFTR trapped in the endoplasmic reticulum). However, after correction of trafficking by 48 h of growth at 27 degrees C, tetrahydrocarbazol and N-phenyltriazine derivatives strongly stimulated Cl(-) conductance with K(d) < 1 microm. The new activators identified here may be useful in defining molecular mechanisms of CFTR activation and as lead compounds in CF drug development.