Activators of the CFTR Cl- channel may be useful for therapy of cystic fibrosis. Short-circuit current (Isc) measurements were done on human bronchial epithelial cells to characterize the best flavone and benzimidazolone CFTR activators identified by lead-based combinatorial synthesis and high-throughput screening. The 7,8-benzoflavone UCcf-029 was a potent activator of Cl- transport, with activating potency (<1 microM) being much better than other flavones, such as apigenin. The benzimidazolone UCcf-853 gave similar Isc but with lower potency (5-20 microM). In combination, the effect induced by maximal UCcf-029 and UCcf-029, UCcf-853, and apigenin increased strongly with increasing basal CFTR activity: for example, Kd for activation by UCcf-029 decreased from >5 to <0.4 microM with increasing basal Isc from approximately 4 microA/cm2 to approximately 12 microA/cm2. This dependence was confirmed in permeabilized Fischer rat thyroid cells stably expressing CFTR. Our results demonstrate efficacy of novel CFTR activators in bronchial epithelia and provide evidence that activating potency depends on basal CFTR activity.