Deletion of Phe-508 (F508) is the most common mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) causing cystic fibrosis. △F508-CFTR has defects in both channel gating and endoplasmic reticulum-to-plasma membrane processing. We identified six novel classes of high affinity potentiators of defective F508-CFTR Cl-channel gating by screening 100,000 diverse small molecules. Compounds were added 15min prior to assay of iodide uptake in epithelial cells co-expressing F508-CFTR and a high sensitivity halide indicator (YFP-H148Q/I152L) in which F508-CFTR was targeted to the plasma membrane by culture at 27℃ for 24h. Thirty-two compounds with submicromolar activating potency were identified; most had tetrahydrobenzothiophene, benzofuran, pyramidinetrione, dihydropyridine, and anthraquinone core structures (360-480 daltons). Further screening of>1000 structural analogs revealed tetrahydrobenzothiophenes that activated F508-CFTR Cl- conductance reversibly with Kd<100nM. Single-cell voltage clamp analysis showed characteristic CFTR currents after F508-CFTR activation. Activation required low concentrations of a cAMP agonist, thus mimicking the normal physiological response. A Bayesian computational model was developed using tetrahydrobenzothiophene structure-activity data, yielding insight into the physical character and structural features of active and inactive potentiators and successfully predicting the activity of structural analogs. Efficient potentiation of defective F508-CFTR gating was also demonstrated in human bronchial epithelial cells from a F508 cystic fibrosis subject after 27℃ temperature rescue. In conjunction with correctors of defective F508-CFTR processing, small molecule potentiators of defective F508-CFTR gating may be useful for therapy of cystic fibrosis caused by the F508 mutation.