Claudins are tight-junction membrane proteins that determine the paracellular permeability to small ions in epithelia. To investigate the structure of the paracellular pore, we have used as a model the MDCK I cell line, which has a high transepithelial resistance, stably transfected with the cation pore-forming claudin, claudin-2, under the control of an inducible promoter. By this means, we can assay the permeability through the claudin pore itself. In this presentation, we describe the characteristics of ion permeation through claudin-2 and its modeling by Brownian dynamics simulation, the identification of an intrapore negatively charged binding site, the mapping of residues in the first extracellular domain by cysteine mutagenesis, and the biochemical status and functional role of two highly conserved extracellular cysteines.