Mutations at many sites within the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel pore region result in changes in chloride conductance. Although chloride binding in the pore - as well as interactions between concurrently bound chloride ions - are thought to be important facets of the chloride permeation mechanism, little is known about the relationship between anion binding and chloride conductance. The present work presents a comprehensive investigation of a number of anion binding properties in different pore mutants with differential effects on chloride conductance. When multiple pore mutants are compared, conductance appears best correlated with the ability of anions to bind to the pore when it is already occupied by chloride ions. In contrast, conductance was not correlated with biophysical measures of anion:anion interactions inside the pore. Although these findings suggest anion binding is required for high conductance, mutations that strengthened anion binding had very little effect on conductance, especially at high chloride concentrations, suggesting that the wild-type CFTR pore is already close to saturated with chloride ions. These results are used to support a revised model of chloride permeation in CFTR in which the overall chloride occupancy of multiple loosely-defined chloride binding sites results in high chloride conductance through the pore. Copyright © 2021 Elsevier B.V. All rights reserved.