Tight junctions (TJs) regulate paracellular permeability across epithelia and vary widely in their transepithelial electrical resistance (TER) and charge selectivity. The claudin family of transmembrane proteins influences these properties. We previously reported that claudin-4 increased TER approximately 300% when expressed in low-resistance Madin-Darby canine kidney (MDCK) II cells and decreased the paracellular permeability for Na(+) more than Cl(-) (Van Itallie C, Rahner C, and Anderson JM. J Clin Invest 107: 1319-1327, 2001). In comparison, we report here that expression of claudin-2 increases TER by only approximately 20% and does not change the ionic selectivity of MDCK II cells from their cation-selective background. To test whether the extracellular domains of claudins-4 and -2 determine their unique paracellular properties, we determined the effects of interchanging these domains between claudins-4 and -2. Inducible expression of wild-type claudins and extracellular domain chimeras increased both the number and depth of fibrils, but the characteristic fibril morphologies of claudin-4 or -2 were not altered by switching extracellular domains. Like claudin-4, chimeras expressing the first or both extracellular domains of claudin-4 on claudin-2 increased TER severalfold and profoundly decreased the permeability of Na(+) relative to Cl(-). In contrast, chimeras expressing the first or both extracellular domains of claudin-2 on claudin-4 increased the TER by only approximately 60 and approximately 40%, respectively, and only modestly altered charge selectivity. These results support a model in which the claudins create paracellular channels and the first extracellular domain is sufficient to determine both paracellular charge selectivity and TER.