Cystic fibrosis transmembrane conductance regulator (CFTR) is an ion channel that couples adenosine triphosphate (ATP) hydrolysis at its nucleotide-binding domains to gating transitions in its transmembrane domains. We previously reported that the charge-neutralized mutant R352C shows two distinct open states, O1 and O2 The two states could be distinguished by their single-channel current amplitudes: O1 having a smaller amplitude (representing a prehydrolytic open state) and O2 having a larger amplitude (representing a post-hydrolytic open state). In this study, a similar phenotype is described for two mutations of another pore-lining residue, N306D and N306E, suggesting that alterations of the net charge within CFTR's pore confer this unique conductance aberration. Because moving either of the two endogenous charges, R303 and R352, to positions further along TM5 and TM6, respectively, also results in this O1O2 phenotype, we conclude that the position of the charged residue in the internal vestibule affects hydrolysis-dependent conductance changes. Furthermore, our data show that the buffer and CFTR blocker morpholino propane sulfonic acid (MOPS-) occludes the O1 state more than it does the O2 state when the net charge of the internal vestibule is unchanged or increased. In contrast, when the net charge in the internal vestibule is decreased, the differential sensitivity to MOPS- block is diminished. We propose a three-state blocking mechanism to explain the charge-dependent sensitivity of prehydrolytic and post-hydrolytic open states to MOPS- block. We further posit that the internal vestibule expands during the O1 to O2 transition so that mutation-induced electrostatic perturbations within the pore are amplified by the smaller internal vestibule of the O1 state and thus result in the O1O2 phenotype and the charge-dependent sensitivity of the two open states to MOPS- block. Our study not only relates the O1O2 phenotype to the charge distribution in CFTR's internal vestibule but also provides a toolbox for mechanistic studies of CFTR gating by ATP hydrolysis.