Glycolytic flux, as well as glucose, fatty acid, and lactate oxidation, was determined in isolated working hearts obtained from 1- and 7-day-old rabbits. One-day-old rabbit hearts were perfused via the inferior cava against a constant aortic and pulmonary arterial afterload, whereas hearts from 7-day-old rabbits were perfused via the left atria against a constant aortic afterload. Hearts were perfused with buffer containing 100 microU/ml insulin and either 1) 11 mM [U-14C/2-3H]glucose, 0.4 mM palmitate, 2 mM lactate; 2) 11 mM glucose, 0.4 mM [1-14C]palmitate, 2 mM lactate; or 3) 11 mM glucose, 0.4 mM palmitate, 2 mM [U-14C]lactate. Glycolytic rates (measured as 3H2O production) were high in 1-day-old hearts but decreased by 7 days (from 2,730 +/- 280 to 580 +/- 80 nmol.min-1.g dry wt-1). Rates of glucose oxidation (measured as 14CO2 production) were lower in both 1- and 7-day-old hearts (59 +/- 4.4 and 23 +/- 2 nmol.min-1.g dry wt-1). Palmitate oxidation rates were low in 1-day-old hearts but dramatically increased by 7 days (22.6 +/- 5.6 and 305 +/- 33 nmol oxidized.min-1.g dry wt-1, respectively). In contrast, lactate was readily oxidized by both 1- and 7-day-old hearts (169 +/- 14 and 456 +/- 52 nmol.min-1.g dry wt-1, respectively). In 1-day-old hearts, 44% of steady-state ATP production from exogenous sources were derived from glycolysis, whereas 18, 13, and 25% were derived from glucose, palmitate, and lactate oxidation, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)