The neonatal proximal tubule has a lower permeability to chloride, higher resistance, and higher relative sodium-to-chloride permeability (P(Na)/P(Cl)) than the adult tubule, which may be due to maturational changes in the tight junction. Claudins are tight-junction proteins between epithelial cells that determine paracellular permeability characteristics of epithelia. We have previously described the presence of two claudin isoforms, claudins 6 and 9, in the neonatal proximal tubule and subsequent reduction of these claudins during postnatal maturation. The question is whether changes in claudin expression are related to changes in functional characteristics in the neonatal tubule. We transfected claudins 6 and 9 into Madin-Darby canine kidney II (MDCK II) cells and performed electrophysiological studies to determine the resultant changes in physiological characteristics of the cells. Expression of claudins 6 and 9 resulted in an increased transepithelial resistance, decreased chloride permeability, and decreased P(Na)/P(Cl) and P(HCO3)/P(Cl). These findings constitute the first characterization of the permeability characteristics of claudins 6 and 9 in a cell model and may explain why the neonatal proximal tubule has lower permeability to chloride and higher resistance than the adult proximal tubule.