Ca(2+) entry through store-operated Ca(2+) release-activated Ca(2+) (CRAC) channels is an essential trigger for lymphocyte activation and proliferation. The recent identification of Orai1 as a key CRAC channel pore subunit paves the way for understanding the molecular basis of Ca(2+) selectivity, ion permeation, and regulation of CRAC channels. Previous Orai1 mutagenesis studies have indicated that a set of conserved acidic amino acids in trans membrane domains I and III and in the I-II loop (E106, E190, D110, D112, D114) are essential for the CRAC channel's high Ca(2+) selectivity. To further dissect the contribution of Orai1 domains important for ion permeation and channel gating, we examined the role of these conserved acidic residues on pore geometry, properties of Ca(2+) block, and channel regulation by Ca(2+). We find that alteration of the acidic residues lowers Ca(2+) selectivity and results in striking increases in Cs(+) permeation. This is likely the result of enlargement of the unusually narrow pore of the CRAC channel, thus relieving steric hindrance for Cs(+) permeation. Ca(2+) binding to the selectivity filter appears to be primarily affected by changes in the apparent on-rate, consistent with a rate-limiting barrier for Ca(2+) binding. Unexpectedly, the mutations diminish Ca(2+)-mediated fast inactivation, a key mode of CRAC channel regulation. The decrease in fast inactivation in the mutant channels correlates with the decrease in Ca(2+) selectivity, increase in Cs(+) permeability, and enlargement of the pore. We propose that the structural elements involved in ion permeation overlap with those involved in the gating of CRAC channels.