Gating of the CFTRCl− channel is associated with ATP hydrolysis at the nucleotide-binding domains (NBD1, NBD2) and requires PKA (protein kinase A) phosphorylation of the R domain. The manner in which the NBD1, NBD2 and R domains of CFTR (cystic fibrosis transmembrane conductance regulator) interact to achieve a properly regulated ion channel is largely unknown. In this study we used bacterially expressed recombinant proteins to examine interactions between these soluble domains of CFTR in vitro. PKA phosphorylated a fusion protein containing NBD1 and R (NBD1–R–GST) on CFTR residues Ser-660, Ser-700, Ser- 712, Ser-737, Ser-768, Ser-795 and Ser-813. Phosphorylation of these serine residues regulated ATP hydrolysis by NBD1–R–GST by increasing the apparent Km for ATP (from 70 to 250 μM) and the Hill coefficient (from 1 to 1.7) without changing the Vmax. When fusion proteins were photolabelled with 8-azido- [α-32P]ATP, PKA phosphorylation increased the apparent kd for nucleotide binding and it caused binding to become co-operative. PKA phosphorylation also resulted in dimerization of NBD1– R–GST but not of R–GST, a related fusion protein lacking the NBD1 domain. Finally, an MBP (maltose-binding protein) fusion protein containing the NBD2 domain (NBD2–MBP) associated with and regulated the ATPase activity of PKA-phosphorylated NBD1–R–GST. Thus when the R domain in NBD1–R–GST is phosphorylated by PKA,ATP binding and hydrolysis becomes cooperative and NBD dimerization occurs. These findings suggest that during the activation of native CFTR, phosphorylation of the R domain by PKA can control the ability of the NBD1 domain to hydrolyse ATP and to interact with other NBD domains.