We examined the activity of ΔF508 cystic fibrosis transmembrane conductance regulator (CFTR) stably expressed in polarized cystic fibrosis bronchial epithelial cells (CFBE41o−) human airway cells and Fisher Rat Thyroid (FRT) cells following treatment with low temperature and a panel of small molecule correctors of ΔF508 CFTR misprocessing. Corr-4a increased ΔF508 CFTR-dependent Cl− conductance in both cell types, whereas treatment with VRT-325 or VRT-640 increased activity only in FRT cells. Total currents stimulated by forskolin and genistein demonstrated similar dose/response effects to Corr-4a treatment in each cell type. When examining the relative contribution of forskolin and genistein to total stimulated current, CFBE41o− cells had smaller forskolin-stimulated Isc following either low temperature or corr-4a treatment (10–30% of the total Isc produced by the combination of both CFTR agonists). In contrast, forskolin consistently contributed greater than 40% of total Isc in ΔF508 CFTR expressing FRT cells corrected with low temperature, and corr-4a treatment preferentially enhanced forskolin dependent currents only in FRT cells (60% of total Isc). ΔF508 CFTR cDNA transcript levels, ΔF508 CFTR C band levels, or cAMP signaling did not account for the reduced forskolin response in CFBE41o− cells. Treatment with non-specific inhibitors of phosphodiesterases (papaverine) or phosphatases (endothall) did not restore ΔF508 CFTR activation by forskolin in CFBE41o− cells, indicating that the Cl− transport defect in airway cells is distal to cAMP or its metabolism. The results identify important differences in ΔF508 CFTR activation in polarizing epithelial models of CF, and have important implications regarding detection of rescued of ΔF508 CFTR in vivo.