The rate of hepatic glucose production (R_a glucose) of rainbow trout (Oncorhynchus mykiss) was measured in vivo by continuous infusion of [6-³H]glucose and in vitro on isolated hepatocytes to examine the role of epinephrine (Epi) in its regulation. By elevating Epi concentration and/or blocking -adrenoreceptors with propranolol (Prop), our goals were to investigate the mechanism for Epi-induced hyperglycemia to determine the possible role played by basal Epi concentration in maintaining resting R_a glucose and to assess indirect effects of Epi in the intact animal. In vivo infusion of Epi caused hyperglycemia (3.75 ± 0.16 to 8.75 ± 0.54 mM) and a twofold increase in R_a glucose (6.57 ± 0.79 to 13.30 ± 1.78 µmol · kg¹ · min¹, n = 7), whereas Prop infusion decreased R_a from 7.65 ± 0.92 to 4.10 ± 0.56 µmol · kg¹ · min¹ (n = 10). Isolated hepatocytes increased glucose production when treated with Epi, and this response was abolished in the presence of Prop. We conclude that Epi-induced trout hyperglycemia is entirely caused by an increase in R_a glucose, because the decrease in the rate of glucose disappearance normally seen in mammals does not occur in trout. Basal circulating levels of Epi are involved in maintaining resting R_a glucose. Epi stimulates in vitro glucose production in a dose-dependent manner, and its effects are mainly mediated by -adrenoreceptors. Isolated trout hepatocytes produce glucose at one-half the basal rate measured in vivo, even when diet, temperature, and body size are standardized, and basal circulating Epi is responsible for part of this discrepancy. The relative increase in R_a glucose after Epi stimulation is similar in vivo and in vitro, suggesting that indirect in vivo effects of Epi, such as changes in hepatic blood flow or in other circulating hormones, do not play an important role in the regulation of glucose production in trout.