Since the initial report by Ussing and Windhager (2) in 1964 of an extracellular pathway for ion flow across frog skin, investigators have speculated about the nature of this route. As the evidence accumulated for such “shunt” paths in a wide variety of epithelia, it was apparent that all shunts were not created equal. The original concept of the paracellular pathway as a nonspecific leak across the tight junction has been replaced with the realization that the selectivity and permeability of tight junctions differ dramatically between tissues. In addition, it has become clear that the permeability of tight junctions can be regulated by a variety of second messengers and physical forces. What remained a mystery until the work of Colegio et al., the current article in focus (Ref. 1, see p. C142 in this issue), was the molecular basis for the differences in ion selectivity of the paracellular pathway. Their elegant work shows that the claudins are the long-sought ionic gatekeepers of tight junctions. Two fundamental factors limited previous investigations of the permeability properties of the paracellular pathway in leaky epithelia: 1) transepithelial measurements are difficult to interpret because they represent an admixture of cellular and paracellular pathways confounded by tissue heterogeneity; and 2) none of the identified molecular components of tight junctions appeared to be directly involved in the determination of paracellular ionic selectivity, although tight junctional structure often was altered when the expression levels of these proteins were manipulated. The first issue, the complexity of interpreting transepithelial measurements, has plagued physiologists for four decades. Measurements of transepithelial electrical resistance in most fluid-transporting epithelia revealed a very high conductance consistent with a substantial paracellular shunt. Investigators observed that the transepithelial resistance could change in either direction when the expression level of putative protein constituents of the tight junction was experimentally increased in cultured epithelia. They concluded that the extent of cell-to-cell adhesion was most likely affected by the protein expression. However, electrical resistance is the sum of all ionic conductances across the entire epithelium, and changes in it do not give insight into the mechanism or site of the change. The transepithelial flux of a neutral tracer molecule such as a sugar alcohol or dextran has been used for decades as a metric of paracellular pore size with the assumption that it could not cross the epithelium by the transcellular route. Size discrimination studies of these neutral species led investigators to conclude that such large holes perforate the tight junctions that discrimination between ionic species seemed unlikely. In contrast, dilution and bi-ionic diffusion potential measurements in many leaky epithelia clearly showed that they were selective for cations or anions. Difficulties in reconciling these apparently conflicting results spawned a variety of speculations about tight-junctional structure and function all directed toward answering the question,“What kind of structure could simultaneously be so leaky and yet so selective?” Colegio et al.(1) convincingly show us that ionic selectivity resides in specific residues in the claudins and that this charge discrimination can be reversed in polarity by substitution of three amino acids in the extracellular domain of claudin-15. They postulate that combinations of the 20 known claudins serve as building blocks in tight junctions and thereby confer tissue-specific ionic permeability properties. Mutations in claudins-14 and-16 are already known to cause two human diseases, and the prospects are bright that …