The molecular determinants for Ca2+ modulation of dihydropyridine (DHP) binding to cardiac Ca2+ channels were identified by mutational neutralization of the glutamate residues that comprise the Ca2+ channel selectivity filter. The binding activity of the DHP (+)-[3H]isradipine, monitored after expression of wild-type and mutant alpha 1 subunits in COS-7 cells, was markedly reduced in four single mutants and a double mutant. Evidence for decreased Ca2+ affinity was obtained for two single mutants in kinetic and equilibrium binding studies. Mutational destabilization of Ca2+ binding resulted in a concomitant decrease of (+)-[3H]isradipine binding affinity. Recovery of (+)-[3H]isradipine binding activity by the allosteric modulator (+)-tetrandrine in two single mutants was associated with a recovery of Ca2+ and DHP binding kinetics to wild-type values. Our findings demonstrate that high-affinity DHP binding is dependent on Ca2+ coordination by glutamate residues which form the selectivity filter of the channel pore.