The effect of recombinant bovine IGF-I (rbIGF-I) on hypo-osmoregulatory ability and the effect of rbIGF-I and cortisol (F) alone and in combination on Na⁺,K⁺-ATPase expression in fresh water (FW) acclimated brown trout (Salmo trutta) were examined in two experiments. In Experiment 1, fish were given three injections of saline or 0.01 or 0.1 µg rbIGF-I/g, respectively, and subjected to a 24-h 25 ppt seawater (SW) challenge test 24 h after the last injection. Fish treated with 0.01 and 0.1 µg rbIGF-I/g had better hypo-osmoregulatory ability than control fish as judged by their higher level of muscle water content and lower plasma osmolality after 24 h exposure to 25 ppt SW. Compared with control fish, gill Na⁺,K⁺-ATPase activity was unchanged 24 h after the first injection at either dose but significantly stimulated after three injections of either dose of rbIGF-I. In Experiment 2, fish were given three injections of saline, 0.1 µg rbIGF-I/g, 4 µg F/g, or 0.1 µg rbIGF-I + 4 µg F/g and sampled in FW 24 h after the last injection. IGF-I and F had additive stimulatory effects on Na⁺,K⁺-ATPase activity and a-subunit Na⁺,K⁺-ATPase mRNA levels in the gill. Injections of IGF-I and F alone and in combination increased Na⁺,K⁺-ATPase-immunoreactive (NKIR) cell number in the primary gill filament but had no effect on secondary lamellar NKIR cell number. NKIR cells were abundant in kidney tubules, pyloric ceca, and posterior intestine, but Na⁺,K⁺-ATPase enzyme activity was unaffected by treatment with F and/or IGF-I in these tissues. F but not rbIGF-I increasedin vitrofluid transport capacity in the posterior intestine. In addition to confirming an overall SW-adaptive effect of rbIGF-I and F in FW-acclimatedS. trutta,the study suggests the effect to be associated with stimulation of chloride cell development and Na⁺,K⁺-ATPase expression in the gill. The study indicates that the stimulatory effects of the two hormones on Na⁺,K⁺-ATPase expression are additive, highly organ specific, and restricted to the primary filament epithelium of the gill.