Octanoate and L-palmitylcarnitine inhibited the synthesis of P-enolpyruvate from alpha-ketoglutarate and malate by isolated guinea pig liver mitochondria. A 50% reduction in P-enolpyruvate formation was obtained with 0.1 to 0.2 mM octanoate or with 0.06 to 0.10 mM L-palmitylcarnitine. At these concentrations, oxidative phosphorylation remained intact and only much higher concentrations of fatty acids altered this process. The addition of NH4Cl in the presence of malate and increasing concentrations of alpha-ketoglutarate (or vice versa) enhanced the formation of glutamate, aspartate, and P-enolpyruvate. The addition of increasing concentrations of NH4Cl in the presence of fixed amounts of malate and alpha-ketoglutarate had a similar effect. Furthermore, the inhibition of P-enolpyruvate synthesis by fatty acids and the reduction of the acetoacetate to beta-hydroxybutyrate ratio were reversed by the addition of NH4Cl. Cycloheximide, which blocks energy transfer at site 1 of the respiratory chain, decreased P-enolpyruvate formation. When cycloheximide and either octanoate or L-palmitylcarnitine were added together, there was an even greater reduction in P-enolpyruvate synthesis from either malate or alpha-ketoglutarate than was noted with either fatty acid alone. Since cycloheximide lowers the rate of ATP synthesis this may in turn reduce P-enolpyruvate formation by a mechanism independent of changes in the mitochondrial NAD+/NADH ratio caused by fatty acids. In the isolated perfused liver metabolizing lactate, the inhibitory effect of octanoate on gluconeogenesis was partially relieved by the addition of 1 mM NH4Cl, but remained unchanged in the presence of 2 mM NH4Cl, despite a highly oxidized NAD+/NADH ratio in the mitochondria. In contrast to glucose synthesis, urea formation was markedly increased during the infusion of 1 mM as well as 2 mM NH4Cl. After cessation of NH4Cl infusion, there was an increase in glucose production, to a rate as high as that observed in the absence of octanoate. This increase was accompanied by the disappearance of alanine, aspartate, and glutamate which had been stored in the liver during NH4Cl infusion. Urea synthesis also decreased progressively. These results indicate that gluconeogenesis in guinea pig liver is regulated, in part, by alterations in the mitochondrial oxidation-reduction state. However, the modulation of this effect by changing the concentrations of intermediates of the aspartate aminotransferase reaction indicates competition for oxalacetate between the aminotransferase reaction and P-enolpyruvate carboxykinase.