This study addresses the mechanisms by which a defect in CFTR impairs pancreatic duct bicarbonate secretion in cystic fibrosis. We used control (PANC‐1) and CFTR‐deficient (CFPAC‐1; ΔF508 mutation) cell lines and measured HCO₃⁻ extrusion by the rate of recovery of intracellular pH after an alkaline load and recorded whole cell membrane currents using patch clamp techniques. 1) In PANC‐1 cells, cAMP causes parallel activation of Cl⁻ channels and of HCO₃⁻ extrusion by DIDS‐sensitive and Na⁺‐independent Cl^‐/HCO₃⁻ exchange, both effects being inhibited by Cl⁻ channel blockers NPPB and glibenclamide. 2) In CFPAC‐1 cells, cAMP fails to stimulate Cl⁻/ HCO₃⁻ exchange and Cl⁻ channels, except after promoting surface expression of ΔF508‐CFTR by glycerol treatment. Instead, raising intracellular Ca²⁺ concentration to 1 μmol/l or stimulating purinergic receptors with ATP (10 and 100 μmol/l) leads to parallel activation of Cl⁻ channels and HCO₃⁻ extrusion. 3) K⁺ channel function is required for coupling cAMP‐ and Ca²⁺‐dependent Cl⁻ channel activation to effective stimulation of Cl⁻/HCO₃⁻ exchange in control and CF cells, respectively. It is concluded that stimulation of pancreatic duct bicarbonate secretion via Cl⁻/ HCO₃⁻ exchange is directly correlated to activation of apical membrane Cl⁻ channels. Reduced bicarbonate secretion in cystic fibrosis results from defective cAMP‐activated Cl⁻ channels. This defect is partially compensated for by an increased sensitivity of CF cells to purinergic stimulation and by alternative activation of Ca²⁺‐dependent Cl⁻ channels, mechanisms of interest with respect to possible treatment of cystic fibrosis and of related chronic pancreatic diseases.—Zsembery, A., Strazzabosco, M., Graf, J. Ca²⁺‐activated Cl⁻ channels can substitute for CFTR in stimulation of pancreatic duct bicarbonate secretion. FASEB J. 14, 2345–2356 (2000)