The CFTR chloride channel is regulated by phosphorylation of its intracellular regulatory (R) domain and ATP binding/hydrolysis at two conserved cytosolic nucleotide binding domains (NBDs). Once phosphorylated by protein kinase A (PKA), gating of the CFTR channel pore in the presence of MgATP displays a bursting pattern. This bursting process reflects an irreversible functional cycle: initiation of a burst coincides with formation of an intramolecular NBD1/NBD2 heterodimer stabilized by two molecules of ATP sandwiched at the dimer interface, while termination of a burst coincides with disruption of this NBD dimer, normally following hydrolysis of one of the two bound nucleotides (Nature 433:876-880). The overall cycle is rate limited by the high enthalpy of the transition state for the pore opening step (J Gen Physiol 128:523-533), but the molecular details of this unstable short-lived structure, and the relative timing of molecular motions that lead from the open-dimer/closed-pore to the tight-dimer/open-pore conformation, are unknown. We present here a thermodynamic study of the opening conformational transition using the rate-equilibrium free energy relationship (REFER) approach. Because REFER analysis provides no information on non-equilibrium systems (J Gen Physiol 134:129-136), we use a CFTR background construct in which mutation of the NBD2 Walker B aspartate prevents ATP hydrolysis, and thus reduces channel gating to a simple equilibrium process. Our results provide direct information on the relative timing of gating motions along the longitudinal axis of the channel protein structure, and on conformational details of the opening transition state.