In airway epithelia, the phosphorylation of nucleoside diphosphate kinase (NDPK) is regulated by [chloride (Cl^–)] and [Na⁺]. NDPK-B is a histidine kinase and has been shown to interact with the cystic fibrosis transmembrane conductance regulator (CFTR) in a cAMP-dependent manner. The aim of this study was to investigate whether the interaction between NDPK-B and CFTR was relevant to the function of CFTR. Cell stimulation with 3-isobutyl-1-methylxanthine (IBMX)/forskolin resulted in the translocation of NDPK-B from the cytosol to the membrane, and Western blot analysis of CFTR immunoprecipitates showed association of NDPK-B and CFTR, which was significantly increased with IBMX/forskolin stimulation. Over-expression of GFP-tagged WT NDPK-B proteins in the human bronchial epithelial cell line 16HBE14o- resulted in a significant increase in the CFTR-mediated whole cell conductance; the outward conductance increased from 241 +/- 148 μS/cm² to 1844 +/- 533 μS/cm² (P < 0.05, unpaired t-test), and inward conductance increased from 259 +/- 193 μS/cm² to 1542 +/- 537 μS/cm² (P< 0.05, unpaired t-test). These interactions are attenuated by a NDPK-B peptide corresponding to amino acids 36 – 54 of the protein. These data suggest that NDPK-B may be important for the signaling processes that regulate CFTR function in epithelial cells.