The effect of fatty acids on the rate of pyruvate decarboxylation was studied in perfused livers from fed rats. The production of 14CO2 from infused [1-14C]pyruvate was employed as a monitor of the flux through the pyruvate dehydrogenase reaction. A correction for other decarboxylation reactions was made using kinetic analyses. Fatty acid (octanoate or oleate) infusion caused a stimulation of pyruvate decarboxylation at pyruvate concentrations in the perfusate below 1 mM (up to 3-fold at 0.05 mM pyruvate) but decreased the rate to one-third of control rates at pyruvate concentrations near 5 mM. These effects were half-maximal at fatty acid concentrations below 0.1 mM. Infusion of 3-hydroxybutyrate also caused a marked stimulation of pyruvate decarboxylation at low pyruvate concentrations. The data suggest that the mechanism by which fatty acids stimulate the flux through the pyruvate dehydrogenase reaction in perfused liver at low (limiting) pyruvate concentrations involves an acceleration of pyruvate transport into the mitochondrial compartment due to an exchange with acetoacetate. Furthermore, it is proposed that a relationship exists between ketogenesis and the regulation of pyruvate oxidation at pyruvate concentrations approximating conditions in vivo.