Cl- secretion by pancreatic duct epithelial cells (PDEC) regulates cellular HCO3- secretion, an important component of the exocrine pancreas. In cystic fibrosis, for example, impaired function of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel results in decreased pancreatic secretion and secondary pancreatic insufficiency. Studies of ion transport by PDEC have been hindered by the lack of a practical in vitro model. We have successfully cultured nontransformed dog PDEC on Vitrogen-coated permeable membranes overlying a feeder layer of myofibroblasts and report the characterization of Cl- channels in these cells. Cl- conductance, assessed through efflux of 125I from PDEC, was stimulated by agents acting via adenosine 3',5'-cyclic monophosphate (cAMP) or cytosolic Ca2+. The Cl- conductances activated by cAMP and Ca2+ were distinct, since they were differentially inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid and, to a lesser extent, by 5-nitro-2-(3-phenylpropylamino)benzoic acid and diphenylamine-2 carboxylate. Patch-clamp studies confirmed the presence of Cl- channels activated by cAMP and Ca2+, with differential inhibition by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. The presence of CFTR Cl- channels in PDEC was confirmed by immunoblotting. These cultured PDEC are an optimal model for studies of pancreatic duct secretion.