The role of cortisol in directing the metabolic response to a combined infusion of glucagon, epinephrine, norepinephrine, and cortisol (stress hormones) was investigated. Chronically catheterized, conscious fasted dogs were studied before hormone infusion and after a 70-hour stress hormone infusion containing glucagon, epinephrine, norepinephrine, and cortisol (n = 11) or containing all these hormones except cortisol (n = 5). Combined stress hormone infusion increased arterial plasma glucagon, cortisol, epinephrine, and norepinephrine approximately sixfold. Whole-body glucose production (Ra), glycogenolysis, and gluconeogenesis were assessed using tracer and arteriovenous-difference techniques. The absence of an increase in cortisol during stress hormone infusion attenuated the increase in arterial plasma glucose concentration and Ra (delta 81 +/- 16 v 24 +/- 3 mg/dL and 1.7 +/- 0.3 v 0.8 +/- 0.4 mg/ kg/min, respectively). However, it did not alter the increase in net hepatic glucose output (delta 0.7 +/- 0.3 v 0.8 +/- 0.4 mg/kg/min). When the increase in cortisol was absent, the increase in net hepatic gluconeogenic precursor uptake was attenuated (delta 0.7 +/- 0.3 v 0.1 +/- 0.3 mg glucose/kg/min) due to a decrease in gluconeogenic precursor levels. The efficiency of gluconeogenesis increased to a greater extent (delta 0.19 +/- 0.07 v 0.31 +/- 0.11) when cortisol was not infused. The absence of an increase in cortisol also led to marked glycogen depletion in the liver (10 +/- 4 v 55 +/- 10 mg/g liver). Cortisol thus plays a pivotal role in the metabolic response to stress hormone infusion by sustaining gluconeogenesis through a stimulatory effect on hepatic gluconeogenic precursor supply and by maintaining hepatic glycogen availability.