In livers from fasted rats perfused with bicarbonate buffer containing bovine albumin and erythrocytes, adrenalectomy decreased glycogen levels and glucose production, impaired the incorporation of 14C from [14C]lactate into glucose or glycogen, and decreased the activity of the active (I) form of glycogen synthase. Cortisol treatment restored gluconeogenesis after 1 h and glycogen synthesis after 2 h. Adrenalectomy did not alter the production of glucose or lactate or the levels of gluconeogenic intermediates in livers from fasted rats perfused with fructose, but reduced the formation of glycogen from this substrate. Adrenalectomy increased the levels of lactate and decreased the levels of P-pyruvate and subsequent intermediates in the gluconeogenic pathway. These changes were reversed by cortisol treatment. It is concluded that glucocorticoids support gluconeogenesis and glycogen synthesis in livers from fasted rats primarily by facilitating a reaction(s) located between pyruvate and P-pyruvate in the gluconeogenic pathway and by promoting the conversion of inactive to active glycogen synthase.