This study investigated whether expression of the common cystic fibrosis transmembrane conductance regulator (CFTR) mutant F508del in the apical membrane of enterocytes confers increased bicarbonate secretory capacity on the intestinal epithelium of F508del mutant mice compared to that of CFTR knockout (KO) mice. CFTR KO mice, F508del mutant mice (F508del) and wild-type (WT) littermates were bred on the FVB/N background. F508del isolated brush border membrane (BBM) contained approximately 5-10% fully glycosylated band C protein compared to WT BBM. Similarly, the forskolin (FSK)-induced, CFTR-dependent short-circuit current (Delta I-sc) of F508del mucosa was approximately 5-10% of WT, whereas the HCO3- secretory response (Delta J(HCO3)(-)) was almost half that of WT in both duodenum and mid-colon studied in vitro and in vivo. While WT intestine retained full FSK-induced in the absence of luminal Cl-, the markedly higher Delta J(HCO3)(-) than Delta I-sc in F508del intestine was dependent on the presence of luminal Cl-, and was blocked by CFTR inhibitors. The Ste20-related proline-alanine-rich kinases (SPAK/OSR1), which are downstream of the with-no-lysine (K) protein kinases (WNK), were rapidly phosphorylated by FSK in WT and F508del, but significantly more slowly in CFTR KO intestine. In conclusion, the data demonstrate that low levels of F508del membrane expression in the intestine of F508del mice significantly increased FSK-induced HCO3- secretion mediated by Cl-/HCO3- exchange. However, in WT mucosa FSK elicited strong SPAK/OSR1 phosphorylation and Cl--independent HCO3- efflux. This suggests that therapeutic strategies which deliver F508del to the apical membrane have the potential to significantly enhance epithelial HCO3- secretion.