Claudin-2 is highly expressed in tight junctions of mouse renal proximal tubules, which possess a leaky epithelium whose unique permeability properties underlie their high rate of NaCl reabsorption. To investigate the role of claudin-2 in paracellular NaCl transport in this nephron segment, we generated knockout mice lacking claudin-2 (Cldn2^−/−). The Cldn2^−/− mice displayed normal appearance, activity, growth, and behavior. Light microscopy revealed no gross histological abnormalities in the Cldn2^−/− kidney. Ultrathin section and freeze-fracture replica electron microscopy revealed that, similar to those of wild types, the proximal tubules of Cldn2^−/− mice were characterized by poorly developed tight junctions with one or two continuous tight junction strands. In contrast, studies in isolated, perfused S2 segments of proximal tubules showed that net transepithelial reabsorption of Na⁺, Cl^–, and water was significantly decreased in Cldn2^−/− mice and that there was an increase in paracellular shunt resistance without affecting the apical or basolateral membrane resistances. Moreover, deletion of claudin-2 caused a loss of cation (Na⁺) selectivity and therefore relative anion (Cl^–) selectivity in the proximal tubule paracellular pathway. With free access to water and food, fractional Na⁺ and Cl^– excretions in Cldn2^−/− mice were similar to those in wild types, but both were greater in Cldn2^−/− mice after i.v. administration of 2% NaCl. We conclude that claudin-2 constitutes leaky and cation (Na⁺)–selective paracellular channels within tight junctions of mouse proximal tubules.