Reconstruction, using non-synaptosomal rat brain mitochondria, has been made here of the malate/oxaloacetate shuttle, made possible by the occurrence in rat brain mitochondria of a carrier mediated transport for oxaloacetate (Km = 60 microM), sensitive to dicarboxylate analogues and mersalyl, and of the two isoenzymes of malate dehydrogenase. Malate/oxaloacetate shuttle shows a high affinity for malate (Km = 100 microM) and Vmax = 40 nmoles NADH oxidized/min X mg mitochondrial protein at 20 degrees C. Its rate appears to depend on the activity of the malate/oxaloacetate exchange across the mitochondrial membrane. A possible role for the oxaloacetate carrier is proposed in aminoacid brain metabolism.