Heterologous expression studies have demonstrated that the PDZ-proteins NHERF1, and NHERF2 modulate CFTR membrane expression, conductivity and interaction with other proteins. To study their biological roles in vivo in an epithelium that highly expresses both, CFTR as well as NHERF1 and NHERF2, we investigated the effect of NHERF1 and 2 ablation, or a combination of the above, on duodenal HCO₃^- secretion in the basal state and after ß₂-adrenergic stimulation and lysophosphatidic acid (LPA) inhibition of CFTR-mediated anion secretion. The proximal duodenum of anesthetized mice was perfused in situ, and HCO₃^- secretion was determined by back-titration. NHERF1 ablation strongly reduced basal and FSK-stimulated HCO₃^- secretory rates, and completely prevented ß₂-adrenergic stimulation. Immunohistochemical staining demonstrated colocalization of the ß₂-adrenergic receptor, CFTR and NHERF1 in the luminal membrane of duodenal crypts and Brunner's glands, which was confirmed by detecting all proteins in Western blots from isolated brush border membranes of murine duodenum. NHERF2 deletion significantly augmented FSK-stimulated HCO₃^- secretion, and prevented the inhibitory effect of LPA on FSK-stimulated HCO₃^- secretion. LPA2 receptors were coexpressed with CFTR and NHERF2 in duodenal crypts, and were localized in the luminal membrane of duodenal enterocytes. The deletion of CFTR abolished agonist-mediated HCO₃^- secretion and any effect of NHERF ablation. We conclude that NHERF1 and 2 differentially modulate basal and agonist-mediated duodenal HCO₃^- secretion in vivo in a CFTR-dependent fashion. NHERF1 is an obligatory linker for ß₂-adrenergic stimulation of CFTR, and strongly augments cAMP-mediated stimulation. NHERF2 confers inhibitory signals, i.e. as a coupling factor between inhibitory LPA receptors and CFTR.