CRAC channels generate Ca(2+) signals critical for the activation of immune cells and exhibit an intriguing pore profile distinguished by extremely high Ca(2+) selectivity, low Cs(+) permeability, and small unitary conductance. To identify the ion conduction pathway and gain insight into the structural bases of these permeation characteristics, we introduced cysteine residues in the CRAC channel pore subunit, Orai1, and probed their accessibility to various thiol-reactive reagents. Our results indicate that the architecture of the ion conduction pathway is characterized by a flexible outer vestibule formed by the TM1-TM2 loop, which leads to a narrow pore flanked by residues of a helical TM1 segment. Residues in TM3, and specifically, E190, a residue considered important for ion selectivity, are not close to the pore. Moreover, the outer vestibule does not significantly contribute to ion selectivity, implying that Ca(2+) selectivity is conferred mainly by E106. The ion conduction pathway is sufficiently narrow along much of its length to permit stable coordination of Cd(2+) by several TM1 residues, which likely explains the slow flux of ions within the restrained geometry of the pore. These results provide a structural framework to understand the unique permeation properties of CRAC channels.