The Calu-3 epithelial cell line expresses the cystic fibrosis transmembrane conductance regulator (CFTR) and is widely used as a model for the acinar cells of human airway submucosal glands. We have examined the ionic basis of cAMP-stimulated fluid secretion by control and CFTR knock-down Calu-3 monolayers cultured at the air-liquid interface. Exposure of control cell monolayers (4.8 cm2) to cpt-cAMP (200 µM) + forskolin (10 µM) evoked fluid secretion of 154 ± 11.8 μl fluid/day, which was reduced to 20 ± 3.6 μl/day in a Calu-3 variant stably expressing short hairpin RNAs complementary to CFTR mRNA. Control secretions contained ~25 mM bicarbonate and became strongly alkaline (pH 9.58 ± 0.14) when equilibrated with room air. Replacing basolateral HCO3- with Hepes and decreasing pCO2 from 5% to 0.035% nearly abolished fluid transport and reduced the pH of secretions to 7.09 ± 0.01. Acetazolamide inhibited fluid secretion by 37% under control conditions and abolished residual secretion under bicarbonate-free conditions, indicating a strict dependence on bicarbonate. Apical glybenclamide (100 μM) and Inh-172 (10 μM) inhibited fluid secretion by 30%, whereas apical DIDS (100 μM) and basolateral bumetanide (10 μM) had no effect. We conclude that sustained, cAMP-stimulated fluid secretion by Calu-3 monolayers involves CFTR but is strictly bicarbonate-dependent. The results are consistent with a model proposed previously for pancreatic ducts(Gray et al., 1988;Novak & Greger, 1988), where apical bicarbonate efflux is mediated by DIDS-insensitive anion exchangers, and CFTR channels mediate chloride recycling rather than transepithelial chloride secretion.