The proximal duodenal epithelium secretes bicarbonate into an adherent mucus layer, thereby protecting the mucosa from injury by gastric acid and pepsin. While bicarbonate secretion is stimulated and inhibited by a number of agonists and antagonists, the apical anion transport pathways have not been addressed fully. The objective was to assess if apical Cl-/HCO3- exchange and Cl-:HCO3- conductance are involved in duodenal mucosal bicarbonate secretion (DMBS). In healthy volunteers, the proximal 4 cm of duodenum was isolated, perfused with either saline or 4,4'-diisothiocyano-2,2'-disulfonic acid (DIDS), and bicarbonate secretion and transepithelial potential difference (PD) were stimulated by either PGE2 or the phosphodiesterase inhibitor theophylline to increase cyclic AMP. Luminal DIDS abolished PGE2-stimulated DMBS, yet had no effect on the increase in PD and failed to significantly alter theophylline-induced DMBS and PD. Therefore, in human proximal duodenum, it appears that PGE2 and cAMP activate distinct HCO3- transport pathways likely involving a DIDS-sensitive Cl-/HCO3- exchanger and DIDS-insensitive HCO3- conductance.