Pancreatic duct cells express a Ca²⁺-activated Cl^- conductance (CaCC), up-regulation of which may be beneficial to patients with cystic fibrosis. Here we report that HPAF, a human pancreatic ductal adenocarcinoma cell line that expresses CaCC, develops into a high-resistance, anion-secreting epithelium. Mucosal ATP (50 µM) caused a 4-fold increase in I_sc, a hyperpolarization of V_t (from -4.9 ± 0.73 mV to -8.5 ± 0.84 mV) and a fall in R_t to less than one-half of resting values. The effects of ATP were inhibited by mucosal niflumic acid (100 µM), implicating an apical CaCC in the response. RT-PCR indicated expression of hClC-2, hClC-3 and hClC-5, but surprisingly not hCLCA-1 or hCLCA-2. K⁺ channel activity was necessary to maintain the ATP-stimulated I_sc . Using a pharmacological approach, we found evidence for two types of K⁺ channels in the mucosal and serosal membranes of HPAF cells, one activated by chlorzoxazone (500 µM) and sensitive to clotrimazole (30 µM), as well as one blocked by clofilium (100 µM) but not chromanol 293B (5 µM). RT-PCR indicated expression of the Ca²⁺ - activated K⁺ channel KCNN4, as well as the acid-sensitive, four transmembrane domain, two pore K⁺ channel, KCNK5 (hTASK-2). Western blot analysis verified the expression of CLC channels, as well as KCNK5. We conclude that HPAF will be a useful model system for studying channels pertinent to anion secretion in human pancreatic duct cells.