The metabolic demands of the newborn heart are met primarily by glucose and lactate. Mitochondria are impermeable to the NADH produced by these cytosolic reactions. The malate/aspanatc and a-glycerophosphate (α-GP) shuttles provide two pathways to transport reducing equivalents into mitochondria. The goals of this study were to compare the capacity of these shuttles in newborn and adult cardiac mitochondria and to measure the maximal activity of the mitochondrial enzymes involved in these shuttles. Shuttle and enzyme capacities were measured in isolated mitochondria from the left and right ventricular free wall of 0–3-d-old and adult pig hearts. Malate/aspartate shuttle capacity was nearly three times greater in the newborn left ventricle compared with adult (newborn, 616 ± 24; adult, 232 ± 28 nmol/min/mg; mean = SEM;n- 8,p< 0.0001). The capacity of the malate/aspartate shuttle of the right ventricular free wall was greater than the left in the adult heart. Despite a decrease in malate/aspartate shuttle capacity, maximal activity of mitochondrial matrix enzymes involved in this pathway were increased in adult mitochondria. α-GP shuttle activity was absent in adult myocardium. Newborn left ventricular myocardium had significant α-GP shuttle activity (44 ± 4 nmol/min/mg) due to enhanced flavin-linked mitochondrial α-GP dehydrogenase activity compated with adult. Interventricular differences in the α-GP shuttle capacity were not found in newborn or adult hearts. These findings suggest a mechanism for the substrate preference of neonatal myocardium.