The thyroid gland in fish mainly secretes the thyroid prohormone 3,5,3',5'-tetraiodothyronine (T₄), and extrathyroidal outer ring deiodination (ORD) of the prohormone to 3,5,3'-triiodothyronine (T₃) is pivotal in thyroid hormone economy. Despite its importance in thyroid hormone metabolism, factors that regulate ORD are still largely unresolved in fish. In addition, the osmoregulatory role of T₃ is still a controversial issue in teleosts. In this study, we investigated the regulation of the ORD pathway by cortisol and T₃ in different organs (liver, kidney, and gills) of Solea senegalensis and the involvement of T₃ in the control of branchial and renal Na(+), K(+)-ATPase activity, a prime determinant of the hydromineral balance in teleosts. Animals were treated with i.p. slow-release coconut oil implants containing cortisol or T₃. Hepatic and renal ORD activities were up-regulated in cortisol-injected animals. T₃-treated fish showed a prominent decrease in plasma-free T₄ levels, whereas ORD activities did not change significantly. Branchial and renal Na(+), K(+)-ATPase activities were virtually unaffected by T₃, but were transiently up-regulated by cortisol. We conclude that cortisol regulates local T₃ bioavailability in S. senegalensis via ORD in an organ-specific manner. Unlike T₃, cortisol appears to be directly implicated in the up-regulation of branchial and renal Na(+), K(+)-ATPase activities.