In view of the occurrence of hepatobiliary disorders in cystic fibrosis (CF) this study addresses the role of the cystic fibrosis transmembrane conductance regulator (CFTR) and of Ca²⁺‐activated Cl⁻ channels in promoting HCO secretion in bile ductular cells. Human cholangiocytes were isolated from control livers and from 1 patient with CF (ΔF508/G542X mutations). Single channel and whole cell currents were analyzed by patch clamp techniques, and HCO secretion was determined by fluorometric analysis of the rate of recovery of intracellular pH following alkaline loading. In control cholangiocytes, both cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) catalytic subunit, activated CFTR Cl⁻ channels that exhibited a nonrectifying conductance of 8 pS and appeared in clusters. Activation of Cl⁻ current by cAMP was associated with an increase in the rate of HCO secretion. The basal rate of HCO secretion was lower in CF than in control cholangiocytes. In both control and CF cholangiocytes, raising intracellular Ca²⁺ concentrations with ionomycin led to a parallel activation of Cl⁻ current and HCO secretion. Consistent with reports that premature stop codon mutations (class I; e.g., G542X) can be read over by treatment with aminoglycoside antibiotics, exposure of CF cholangiocytes to gentamicin restored activation by cAMP of Cl⁻ current and HCO secretion. The observation that activation of Ca²⁺‐dependent Cl⁻ channels can substitute for cystic fibrosis transmembrane conductance regulator (CFTR) in supporting HCO secretion and the efficacy of gentamicin in restoring CFTR function and HCO secretion in class I mutations are of potential clinical interest.