We have previously described a protocol for the simultaneous isolation and reconstitution of a protein kinase A (PKA)-sensitive outwardly rectified chloride channel (ORCC) and the cystic fibrosis transmembrane conductance regulator (CFTR) from bovine tracheal epithelium. Immunoprecipitation of CFTR from this preparation prevented PKA activation of the ORCC, suggesting that CFTR regulated the ORCC and that this regulatory relationship was preserved throughout the purification procedure. We now report the purification of CFTR from bovine tracheal epithelia and the purification of a CFTR conduction mutant (G551D CFTR) from retrovirally transduced mouse L cells using a combination of alkali stripping, Triton-X extraction, and immunoaffinity chromatography. Immunopurified CFTR proteins were reconstituted in the absence and presence of ORCC. To test the hypothesis that only functional CFTR can support activation of ORCC by PKA and ATP, we used an inhibitory anti-CFTR505-511 peptide antibody or G551D CFTR. When anti- CFTR505-511 peptide antibodies were present prior to the addition of PKA and ATP, activation of both the ORCC and CFTR was prevented. If the antibody was added after activation of the ORCC and CFTR Cl- channels by PKA and ATP, only the CFTR Cl- channel was inhibited. When ORCC and G551D CFTR were co-incorporated into planar bilayers, only the ORCC was recorded and this channel could not he further activated by the addition of PKA and ATP. Thus, functional CFTR is required for activation of the ORCC by PKA and ATP. We also tested the hypothesis that PKA activation of ORCC was dependent on the extracellular presence of ATP. We added ATP on the presumed extracellular side of the lipid bilayer under conditions where it was not possible to activate the ORCC, i.e. in the presence of inhibitory anti-CFTR505-511 antibody or G551D CFTR. In both cases the ORCC regained PKA sensitivity. Moreover, the addition of hexokinase + glucose to the extracellular side prevented activation of the ORCCs by PKA and ATP in the presence of CFTR. These experiments confirm that both the presence of CFTR as well as the presence of ATP on the extracellular side is required for activation of the ORCC by PKA and ATP.