Two experiments are described in which the interaction of prolactin, cortisol and growth hormone on hypoosmoregulation in the anadromous brown trout was studied. In experiment 1, fish at the postsmolt stage in freshwater (FW) were given four injections on alternate days of 5 μg cortisol (F)/g in combination with 0, 0.2, 1 or 2 μg ovine prolactin (oPRL)/g. Additional groups received 2 μg oPRL/g or saline as control. In experiment 2, FW parr were given four injections on alternate days with 5 μg F/g and 2 μg ovine growth hormone (oGH)/g in combination with 0, 0.2, 1 or 2 μg oPRL/g. Additional groups received 2 μg oPRL/g, 2 μg oGH/g, 5 μg F/g or saline as control. In both experiments, the fish were subjected to a 48 hr seawater (SW) challenge test 24 hr after the last injection. Muscle water content, plasma osmolality and ion levels, kidney and gill Na , K -ATPase activity were measured. In experiment 1, F-treated fish had better hypoosmoregulatory capacity than control fish as judged by a higher level of muscle water content and lower plasma osmolality after 48 hr in SW. All three doses of oPRL completely abolished this action of F. Gill Na ,K -ATPase activity was stimulated by F and unaffected by oPRL at any dose. In experiment 2, oPRL impaired, whereas F and oGH (injected individually or together) improved performance in the 48 hr SW test relative to control fish, judged by plasma osmolality and muscle water content. Ovine PRL inhibited the combined action of F and oGH in a dose-related manner but could not completely counteract the combined effect of these hormones. F and oGH had additive stimulatory effects on gill Na ,K -ATPase activity. This activation was counteracted by coinjection of oPRL in a dose-related manner but not below the level found in the F-treated fish. We conclude that exogenous oPRL inhibits the hypoosmoregulatory action of F. However, oPRL does not antagonize the F-dependent stimulation of gill Na ,K -ATPase activity. The observed antagonism between PRL and F in SW-acclimation may therefore occur on a target different from gill Na ,K -ATPase.