The mechanism of Cl ion permeation through single cystic fibrosis transmembrane conductance regulator (CFTR) channels was studied using the channel-blocking ion gluconate. High concentrations of intracellular gluconate ions cause a rapid, voltage-dependent block of CFTR Cl channels by binding to a site approximately 40% of the way through the transmembrane electric field. The affinity of gluconate block was influenced by both intracellular and extracellular Cl concentration. Increasing extracellular Cl concentration reduced intracellular gluconate affinity, suggesting that a repulsive interaction occurs between Cl and gluconate ions within the channel pore, an effect that would require the pore to be capable of holding more than one ion simultaneously. This effect of extracellular Cl is not shared by extracellular gluconate ions, suggesting that gluconate is unable to enter the pore from the outside. Increasing the intracellular Cl concentration also reduced the affinity of intracellular gluconate block, consistent with competition between intracellular Cl and gluconate ions for a common binding site in the pore. Based on this evidence that CFTR is a multi-ion pore, we have analyzed Cl permeation and gluconate block using discrete-state models with multiple occupancy. Both two- and three-site models were able to reproduce all of the experimental data with similar accuracy, including the dependence of blocker affinity on external Cl (but not gluconate) ions and the dependence of channel conductance on Cl concentration. The three-site model was also able to predict block by internal and external thiocyanate (SCN) ions and anomalous mole fraction behavior seen in Cl/SCN mixtures.