Recent research has shown that nitric oxide (NO) produced by nitric oxide synthases (NOS) is an inhibitor of ion transporter activity and a modulator of epithelial ion transport in fish, but little is known on changes in the NOS/NO system during osmotic stress. We hypothesized that the NOS/NO system responds to salinity changes as an integrated part of the acclimation process. Expression and localization of nos1/Nos1 and nos2/Nos2 were investigated in gill, kidney, and intestine of freshwater (FW)- and seawater (SW)-transferred trout using quantitative PCR, Western blotting, and immunohistochemistry, along with expressional changes of major ion transporters in the gill. The classical branchial ion transporters showed expected expressional changes upon SW transfer, there among a rapid decrease in Slc26a6 mRNA, coding a branchial Cl /Formula: see text exchanger. There was a major downregulation of nos1/ nos2/Nos2 expression in the gill during SW acclimation. A significant decrease in plasma nitrite supported an overall decreased Nos activity and NO production. In the middle intestine, Nos1 was upregulated during SW acclimation, whereas no changes in nos/Nos expression were observed in the posterior intestine and the kidney. Nos1 was localized along the longitudinal axis of the gill filament, beneath smooth muscle fibers of the intestine wall and in blood vessel walls of the kidney. Nos2 was localized within the epithelium adjacent to the gill filament axis and in hematopoietic tissues of the kidney. We conclude that downregulation of branchial NOS is integrated to the SW acclimation process likely to avoid the inhibitory effects of NO on active ion extrusion.