VOLTAGE-GATED Ca²⁺ channels link changes in membrane potential to the delivery of Ca²⁺, a key second messenger for many cellular responses¹. Ca²⁺ channels show selectivity for Ca²⁺ over more plentiful ions such as Na⁺ or K⁺ by virtue of their high-affinity binding of Ca²⁺ within the pore^{2& ndash;6}. It has been suggested that this binding involves four conserved glutamate residues^{7& ndash;10} in equivalent positions in the putative pore-lining regions of repeats I& ndash;IV in the Ca²⁺ channel & alpha;₁ subunit. We have carried out a systematic series of single amino-acid substitutions in each of these positions and find that all four glutamates participate in high-affinity binding of Ca²⁺ or Cdd²⁺. Each glutamate carboxylate makes a distinct contribution to ion binding, with the carboxylate in repeat III having the strongest effect. Some single glutamate-to-lysine mutations completely abolish micromolar Ca²⁺ block, indicating that the pore does not possess any high-affinity binding site that acts independently of the four glutamate residues. The prevailing model of Ca²⁺permeation^2,3 must thus be modified to allow binding of two Ca²⁺ ions in close proximity^11,12, within the sphere of influence of the four glutamates. The functional inequality of the glutamates may be advantageous in allowing simultaneous interactions with multiple Ca²⁺ ions moving single-file within the pore. Competition among Ca²⁺ ions for individual glutamates^11,12, together with repulsive ion-ion electrostatic interaction^2,3, may help achieve rapid flux rates through the channel^{2& ndash;5}.