The role of the cellular redox state in the hormonal stimulation of gluconeogenesis was studied in hemoglobin-free perfused rat liver, by fluorimetric measurement of the redox states of intracellular pyridine nucleotides. The maximum rate of glucose production from lactate/pyruvate mixture was observed with a lactate/pyruvate ratio of 10/1, which corresponds to the ratio observed in vivo. Increased reduction of pyridine nucleotides on infusion of ethanol or octanoate was associated with an increased production of glucose from pyruvate, whereas glucose production from lactate decreased. Stimulation of gluconeogenesis from lactate by glucagon was affected by the lactate/pyruvate ratio; a decrease of the lactate/pyruvate ratio resulted in a decrease of the efficacy of glucagon. Stimulation by glucagon of glucose production from pyruvate was abolished during octanoate infusion, although it was still observable during ethanol infusion. In contrast to glucagon, the stimulatory effect of norepinephrine on gluconeogenesis was unaffected by the ratio of lactate to pyruvate. Norepinephrine in the presence of octanoate and ethanol still induced stimulation of glucose production from lactate and pyruvate, which was always accompanied by a transient reduction of pyridine nucleotides. The results demonstrate that the regeneration of NADH in the cytosol is one of the regulatory factors in gluconeogenesis, and that the effects of glucagon and norepinephrine on gluconeogenesis and on the redox state of pyridine nucleotides are not identical.