Cystic fibrosis (CF) is characterised by impaired epithelial ion transport and is caused by mutations in the cystic fibrosis conductance regulator protein (CFTR), a cAMP/PKA and ATP-regulated chloride channel. We recently demonstrated a cAMP/PKA/calcineurin (CnA)-driven association between annexin 2 (anx 2), its cognate partner -S100A10 and cell surface CFTR. The complex is required for CFTR and outwardly rectifying chloride channel function in epithelia. Since the cAMP/PKA-induced Cl(-) current is absent in CF epithelia, we hypothesized that the anx 2-S100A10/CFTR complex may be defective in CFBE41o cells expressing the commonest F508del-CFTR (DeltaF-CFTR) mutation. Here, we demonstrate that, despite the presence of cell surface DeltaF-CFTR, cAMP/PKA fails to induce anx 2-S100A10/CFTR complex formation in CFBE41o- cells homozygous for F508del-CFTR. Mechanistically, PKA-dependent serine phosphorylation of CnA, CnA-anx 2 complex formation and CnA-dependent dephosphorylation of anx 2 are all defective in CFBE41o- cells. Immunohistochemical analysis confirms an abnormal cellular distribution of anx 2 in human and CF mouse epithelia. Thus, we demonstrate that cAMP/PKA/CnA signaling pathway is defective in CF cells and suggest that loss of anx 2-S100A10/CFTR complex formation may contribute to defective cAMP/PKA-dependent CFTR channel function.