Claudins are integral membrane proteins at the tight junction that determine the paracellular permeability properties of epithelial barriers. Claudins have four transmembrane domains and two extracellular domains. The first extracellular domain has been proposed to form the lining of the paracellular pore. We have developed an in vitro model, using inducible overexpression of claudin-2 in MDCK I cells, to measure the conductance of a claudin ion pore. The claudin-2 pore is narrow, cation-selective, and behaves as if it has a strong, negatively charged intrapore binding site. By site-directed mutagenesis, we have identified an acidic residue in the first extracellular domain (D65) as the intrapore Na+-binding site. We have also performed cysteine mutagenesis of selected residues in the first extracellular domain, and assessed their accessibility to membrane-impermeant thiol-reactive reagents. These studies have allowed us to infer that I66 is located within the pore lumen, Y35 is located outside the pore vestibule exposed extracellularly, and D65 is in close proximity to an intermolecular interface.