Heterologous expression studies have demonstrated that the PDZ-proteins NHERF1, NHERF2 and PDZK1 (NHERF3) 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 and all three NHERF proteins, we investigated the effect of NHERF1, 2 and PDZK1 ablation, or a combination of the above, on duodenal HCO₃^- secretion in the basal state and after agonist and inhibitor application. The proximal duodenum of anesthetized mice was perfused in situ, and HCO₃^- secretion was determined by back-titration. NHERF1 ablation strongly reduced basal and forskolin (FSK)-stimulated HCO₃^- secretory rates, and completely prevented b2-adrenergic stimulation. NHERF2 deletion significantly augmented FSK-stimulated HCO₃^- secretion, prevented the inhibitory effect of LPA, and partially rescued the suppressed basal HCO₃^- secretion resulting from NHERF1 ablation. PDZK1 ablation reduced basal HCO₃^- secretion but not the response to FSK. The deletion of CFTR abolished agonist-mediated HCO₃^- secretion and any effect of NHERF ablation. We conclude that the three NHERF proteins differentially modulate basal and agonist-mediated duodenal HCO₃^- secretion in vivo in a CFTR- dependent fashion. NHERF1 is an obligatory linker for b2-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.