Anion and fluid secretion are both defective in cystic fibrosis (CF); however, the transport mechanisms are not well understood. In this study, Cl- and HCO3- secretion was measured using genetically matched CF transmembrane conductance regulator (CFTR)-deficient and CFTR-expressing cell lines derived from the human airway epithelial cell line Calu-3. Forskolin stimulated the short-circuit current (Isc) across voltage-clamped monolayers, and also increased the equivalent short-circuit current (Ieq) calculated under open-circuit conditions. Isc was equivalent to the HCO3- net flux measured using the pH-stat technique, whereas Ieq was the sum of the Cl- and HCO3- net fluxes. Ieq and HCO3- fluxes were increased by bafilomycin and ZnCl2, suggesting that some secreted HCO3- is neutralized by parallel electrogenic H+ secretion. Ieq and fluid secretion were dependent on the presence of both Na+ and HCO3-. The carbonic anhydrase inhibitor acetazolamide abolished forskolin stimulation of Ieq and HCO3- secretion, suggesting that HCO3- transport under these conditions requires catalysed synthesis of carbonic acid. Cl- was the predominant anion in secretions under all conditions studied and thus drives most of the fluid transport. Nevertheless, 50-70% of Cl- and fluid transport was bumetanide-insensitive, suggesting basolateral Cl- loading by a sodium-potassium-chloride cotransporter 1 (NKCC1)-independent mechanism. Imposing a transepithelial HCO3- gradient across basolaterally permeabilized Calu-3 cells sustained a forskolin-stimulated current, which was sensitive to CFTR inhibitors and drastically reduced in CFTR-deficient cells. Net HCO3- secretion was increased by bilateral Cl- removal and therefore did not require apical Cl-/HCO3- exchange. The results suggest a model in which most HCO3- is recycled basolaterally by exchange with Cl-, and the resulting HCO3--dependent Cl- transport provides an osmotic driving force for fluid secretion.