Inhibition of endogenous long chain fatty acids oxidation by tetradecylglycidate (TDGA) impeded gluconeogenesis from lactate or from low concentrations of pyruvate (<0.5 mm). The inhibitory effect of TDGA was overcome by medium and short chain fatty acid or by concentrations of pyruvate about 0.5 mm, but not by 10-fold higher concentrations of lactate. Despite decreased energy demand when gluconeogenesis was inhibited by TDGA, the pyruvate-induced increase in hepatic oxygen consumption was similar to the control, indicating that pyruvate transport across the mitochondrial membrane and/or its decarboxylation was not altered, and therefore can not be responsible for the inhibition of gluconeogenesis. Neither does a deficiency of acetyl-CoA explain the decrease in the gluconeogenic flux since high pyruvate loads (>0.5 mm), β-hydroxybutyrate or even ethanol was capable of overcoming the inhibitory effect of TDGA in the absence of significant changes in the hepatic content of acetyl-CoA. At low (<0.3 mm), presumably physiological, pyruvate concentrations, its rate of mitochondrial utilization is limited by the activity of the monocarboxylate transporter. Agents that reduced the mitochondrial NAD system, and therefore reduced flux through pyruvate dehydrogenase, like β-hydroxybutyrate or ethanol, stimulated gluconeogenesis when fatty acid oxidation was inhibited. The latter observations indicate that the primary role of endogenous fatty acid, when substrate availability is limiting, is to spare mitochondrial pyruvate by decreasing its oxidation, and therefore shifting the partitioning between the carboxylation and decarboxylation reactions toward the former.