The objective of this study was to examine if RU486, a corticosteroid antagonist, modified hepatocyte metabolism and blocked the metabolic effects of exogenously administered cortisol in rainbow trout (Oncorhynchus mykiss). The fish were given a single intraperitoneal implant of either coconut oil alone or coconut oil containing RU486 (100 mg.kg-1), cortisol (100 mg.kg-1), or a combination of RU486 and cortisol and were sampled 7 days postimplantation. The RU486 implants had no effect on plasma cortisol and glucose concentrations, hepatocyte glycogen content, and total glucose production, but increased in vitro hepatocyte glycogen breakdown. Cortisol implantation had no effect on plasma glucose concentration, hepatocyte glycogen content, glycogen breakdown, or total glucose production, while it significantly increased alanine oxidation and gluconeogenesis in rainbow trout hepatocytes. Hepatocyte responsiveness to epinephrine and glucagon stimulation of total glucose production was not modified by either RU486 or cortisol treatment, whereas the insulin response on total glucose production was decreased with cortisol. RU486 treatment modified hepatocyte glycogen metabolism and blocked the cortisol-induced increases in alanine gluconeogenesis and glycogen mobilization for endogenous use by the hepatocytes. These results indicate that cortisol enhances the metabolic potential of hepatocytes, thereby adapting the fish to cope with stress. This study provides further validation of RU486 as a tool for studying metabolic actions of cortisol in fish.