Chloride impermeability of epithelial cells can account for many of the experimental and clinical manifestations of cystic fibrosis (CF)1,2. Activation of apical-membrane Cl− channels by cyclic AMP-mediated stimuli is defective in CF airway epithelial cells3,4, despite normal agonist-induced increases in cellular cAMP levels4,5. This defect in Cl− channel regulation has been localized to the apical membrane by exposing the cytoplasmic surface of excised membrane patches to the catalytic subunit (C subunit) of cAMP-dependent protein kinase and ATP. In membranes from normal cells, C-subunit activated Cl− channels with properties identical to those stimulated by cAMP-dependent agonists during cell-attached recording. Activation by the C subunit was not observed in CF membranes, but the presence of Cl− channels was verified by voltage-induced activation. The failure of the C subunit to activate the Cl− channels of CF membranes indicates that the block in their cAMP-mediated activation lies distal to induction of cAMP-dependent protein kinase activity and focuses our attention on the Cl− channel and its membrane-associated regulatory proteins as the probable site of the CF defect.