While studies clearly point to a role for cortisol signaling in seawater adaptation, very little is known about salinity impact on glucocorticoid receptor (GR) expression in fish. To this end, we investigated the temporal GR expression in the gill and liver of rainbow trout (Oncorhynchus mykiss) to salinity exposure. Trout were subjected to gradual salinity increases (11 ppt for 1 d, 17 ppt for 2 d and 23 ppt for 2 d) over a five day period. Gill Na(+), K(+)-ATPase alpha-subunit mRNA showed a transient elevation with salinity exposure, while gill cystic fibrosis transmembrane conductance regulator mRNA was not significantly affected by salinity. Liver PEPCK transcript levels showed a transient increase at day 1, but not at day 3 or day 5 of salinity exposure, while the activity of this enzyme was significantly depressed at all time points. Liver glycogen content was also significantly reduced by salinity exposure compared to the freshwater group. Gill GR transcript levels were 3-fold greater upon salinity exposure and this level was maintained over the 5 day period, while gill GR protein content remained unchanged except for a significant drop at day 1 of salinity exposure. Liver GR transcript levels showed no significant change with salinity exposure, while GR protein content was transiently elevated at day 3, but not at day 1 or day 5 of salinity exposure. The tissue-specific GR transcript response in the gill leads us to hypothesize a role for osmosensory signal transduction pathway in the regulation of GR expression in fish. Collectively, salinity exposure modulates GR expression and glucocorticoid signaling in rainbow trout.