Immediately after birth, glycolysis and lactate oxidation are the major source of ATP production in the rabbit heart. Although the ability of heart to oxidize fatty acid increases within days, glucose oxidation rates remain low in the first week after birth. To further examine the changes in energy substrate use in the newborn period, we developed a right and left ventricular isolated working heart model, in which 2-wk-old rabbit hearts were perfused with buffer containing 11 mM glucose, 0.8 mM palmitate, 0.5 mM lactate, and 100 μU/mL insulin. Hearts were perfused at a 7.5 mm Hg left atrial preload, a 4.5 mm Hg superior vena cava preload, a 30 mm Hg aortic afterload, and a 4.5 mm Hg pulmonary artery afterload. Glycolytic rates [measured as 3H2O production from (5−3H)-glucose] were 791 ± 108 nmol/g dry weight min−1 (mean ± SEM). Oxidation rates of glucose, lactate, and palmitate (measured as 14CO2 production from 14C substrates) were 94 ± 15, 126 ± 13, and 60 ± 8 nmol/g dry weight-min−1, respectively. In these hearts, the majority of ATP production derived from exogenous sources was obtained from fatty acid oxidation (52%), whereas 11, 23, and 15% of ATP requirements were derived from glycolysis, glucose oxidation, and lactate oxidation, respectively. These studies demonstrate that by 2 wks of age in rabbits, fatty acids are the major source of energy in the heart. However, although the contribution of glucose oxidation to ATP production has increased compared with 1− or 7-d-old rabbit hearts, glucose oxidation rates are still low compared with adult hearts.