It is widely believed that the selectivity of voltage-dependent calcium channels is mainly controlled by amino acid residues contained within four p-loop motifs forming the pore of the channel. An examination of the amino acid sequences of high voltage-activated calcium channels reveals that their domain III S5-H5 regions contain a highly conserved motif with homology to known EF hand calcium binding proteins, hinting that this region may contribute to channel permeation. To test this hypothesis, we used site-directed mutagenesis to replace three conserved negatively charged residues in the N-type calcium channel alpha1B subunit (Glu-1321, Asp-1323, and Glu-1332) with positively charged amino acids (lysine and arginine) and studied their effect on ion selectivity using whole cell and single channel patch clamp recordings. Whereas the wild type channels conducted barium much more effectively than calcium, the mutant displayed nearly equal permeabilities for these two ions. Individual replacement of residue 1332 or a double substitution of residues 1321 and 1323 with lysine and arginine, respectively, were equally effective. Disruption of the putative EF hand motif through replacement of the central glycine residue (1326) with proline resulted in a similar effect, indicating that the responses observed with the triple mutant were not due to changes in the net charge of the channel. Overall, our data indicate that residues outside of the narrow region of the pore have the propensity to contribute to calcium channel permeation. They also raise the possibility that interactions of calcium ions with a putative calcium binding domain at the extracellular side of the channel may underlie the differential permeabilities of the channel for barium and calcium ions.