Cystic fibrosis transmembrane conductance regulator (CFTR) is a non-rectifying, low-conductance channel regulated by ATP and phosphorylation, which mediates apical chloride conductance in secretory epithelia and malfunctions in cystic fibrosis (CF). Mutations at Lys 335 and Arg 347 in the sixth predicted transmembrane helix of CFTR alter its halide selectivity in whole-cell studies and its single channel conductance, but the physical basis of these alterations is unknown and permeation in CFTR is poorly understood. Here we present evidence that wild-type CFTR can contain more than one anion simultaneously. The conductance of CFTR passes through a minimum when channels are bathed in mixtures of two permeant anions. This anomalous mole fraction effect can be abolished by replacing Arg 347 with an aspartate and can be toggled on or off by varying the pH after the same residue is replaced with a histidine. Thus the CFTR channel should provide a convenient model in which to study multi-ion pore behaviour and conduction. The loss of multiple occupancy may explain how naturally occurring CF mutations at this site cause disease.