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Claudins are a large family of integral transmembrane tight junction (TJ) proteins involved in regulating the permeability of the paracellular pathway. In these studies, we clone and describe the tissue distribution of four claudin-3 genes (designated Tncldn3a, Tncldn3b, Tncldn3c, and Tncldn3d) from the euryhaline spotted green puffer fish Tetraodon nigroviridis and examine the response of Tetraodon and Tncldn3 mRNAs to salinity variation (freshwater, FW; seawater, SW; and hypersaline seawater, HSW). In Tetraodon, genes encoding for claudin-3 TJ proteins are widely expressed, suggesting that claudin-3 proteins participate in regulating paracellular permeability across various epithelia within fishes. Of particular note is the widespread distribution of Tncldn3 genes in tissues that regulate hydromineral balance (gills, skin, kidney, and intestine). Renal and intestinal tissues express all four Tncldn3 genes, while the gills and skin specifically express Tncldn3a and Tncldn3c. In response to salinity variation, Tetraodon exhibits characteristics typical of a euryhaline fish species: moderate changes in blood osmolality and muscle moisture content; alterations in gill, kidney, and intestinal Na(+)-K(+)-ATPase activity; and unaltered Na(+)-K(+)-ATPase activity in the integument. In conjunction with these changes, Tncldn3 mRNA expression exhibits marked and significant salinity-dependent alterations that are both tissue and gene specific. Overall, our data suggest that a decreased abundance of claudin-3 in Tetraodon occurs in "leakier" epithelia and that claudin-3 TJ proteins will likely play an important role in the maintenance of hydromineral balance across osmoregulatory epithelia of euryhaline fishes.