A 2-factorial (3x3) injection experiment was used to investigate the effect and interaction between different hormones on the initial phase of seawater (SW) acclimation in brown trout (Salmo trutta). Each fish was given 4 injections on alternate days in freshwater (FW). Factor 1 was either saline, 2 micrograms ovine prolactin (oPRL)/g, or 2 micrograms ovine growth hormone (oGH)/g. Factor 2 was either 0, 0. 01, or 0.1 mirograms recombinant human insulin-like growth factor-I (rhIGF-I)/g. In each of the 9 treatment groups, half of the fish were subjected to a 48-h SW-challenge test, and the remaining fish were sham-transferred to FW one day after the last injection. Hypo-osmoregulatory performance was increased by GH and impaired by PRL treatment as judged by changes in plasma osmolality, [Na+], [Cl-], total [Mg] and muscle water content (MWC) after SW transfer. IGF-I reduced plasma osmolality after transfer to SW but had no effect on plasma total [Mg] or MWC. The effects of the two factors on plasma osmolality, [Na+], [Cl-], and MWC were additive. In sham-transferred fish, GH and IGF-I, alone and in combination, stimulated Na+,K+-ATPase alpha-subunit mRNA (alpha-mRNA) content in the gill. This was paralleled by an overall increase in gill Na+, K+-ATPase activity in fish treated with 0.01 micrograms IGF-I/g. Simultaneous administration of PRL completely inhibited the increase in gill alpha-mRNA observed in the IGF-I-injected groups. Combination of GH and IGF-I did not further affect the alpha-mRNA level relative to the single hormone-injected groups. There was an overall decrease in Na+,K+-ATPase activity in pyloric caeca and middle intestine by the low dose and both doses of IGF-I respectively. No effect was observed in the posterior intestine. PRL and GH treatments did not affect enzyme activity in any intestinal segment. Both doses of IGF-I increased Na+,K+-ATPase-immunoreactive (NKIR) cell density in gill primary filaments. PRL and GH had no effect on primary filament NKIR cell density. GH and both doses of IGF-I reduced secondary lamellar NKIR cell density, whereas PRL had no effect. The main conclusion is that IGF-I and GH induce an overall redistribution of NKIR cells away from the secondary lamella onto the primary filament of FWacclimated trout. This is associated with an overall increased alpha-mRNA level in the gill, which may reflect an increased expression within individual NKIR cells in the primary filament. PRL completely abolished the IGF-I stimulation of alpha-mRNA levels, suggesting a desensitisation of the gill tissue to IGF-I, which may explain the overall anti-SW adaptive effect of PRL.