In the possum (Trichosurus vulpecula) ileum the prostaglandin E₂ (PGE₂)-stimulated short circuit current (Isc) is dependent upon serosal Na⁺ and HCO₃^– and inhibited by serosal 4,4’-diisothiocyano-stilbene-2,2’-disulfonate (DIDS) (Bartolo et al. 2009a), but insensitive to bumetanide and independent of Cl^– (Bartolo et al. 2009b). This suggests that in this tissue secretagogues stimulate electrogenic HCO₃^– secretion. Here we have used the pH stat technique to determine the serosal to mucosal flux of HCO₃^– (J^sm_HCO3) in short-circuited tissues and immunohistochemistry to identify the potential transporters involved in the secretory response. Animals were euthanased by an intracardiac injection of barbiturate administered under halothane-induced anesthesia. Ileal tissue was collected and either stripped of the underlying connective tissues and mounted in the Ussing chamber for pH stat studies or fixed for immunohistochemistry. In NaCl Ringer’s, the spontaneous short-circuit current (Isc) was 3.0 ± 0.5 µEq.cm^-2.h^-1 and the J^sm_HCO3 was 5.6 ± 0.9 µM.cm^-2.h^-1 (All values X±SEM., n=8). Replacement of all but 20 mM of the Ringer’s Cl^– with gluconate had little effect on the Isc (3.0 ± 0.3 µEq.cm^-2.h^-1) but reduced J^sm_HCO3 to 0.4 ± 0.1 µM.cm^-2.h^-1 and total replacement of Cl^– with gluconate completely inhibited J^sm_HCO3 (0.1 ± 0.1 µM.cm^-2.h^-1). In low NaCl Ringer’s PGE₂ (1µM serosal) stimulated the Isc and J^sm_HCO3 by 1.6 ± 0.2 µEq.cm^-2.h^-1 and 1.0 ± 0.2 µM.cm^-2.h^-1, respectively, significantly greater (P<0.05, Student’s t test) than the change in Isc (0.1±0.1 µEq.cm^-2.h^-1) and J^sm_HCO3 (0.2±0.2 µM.cm^-2.h^-1) in unstimulated control tissues. The PGE₂-stimulated Isc was inhibited by mucosal N-(2-Naphthalenyl)-((3,5-dibromo-2,4-dihydroxyphenyl)methylene) glycine hydrazide (IC₅₀ = 22µM) and 5 nitro-2-(3-phenylpropylamino) benzoic acid (IC₅₀ = 29µM), but not 1 mM DIDS. Immunoreactivity for the cystic fibrosis transmembrane conductance regulator (CFTR) was evident in the apical membrane of cells in the crypts and lower villi of the ileal epithelium, whereas the Cl/HCO₃ exchangers, SLC26A3 and A6, where present in the apical membrane of the villous cells. These data indicate that in the possum ileum both electroneutral and electrogenic HCO₃^– secretion occur and HCO₃^– secretion accounts for the bulk of the PGE₂-stimulated Isc, which is most likely mediated by CFTR.