Isoproterenol increases phosphorylation of LKB, 5'-AMP-activated protein kinase (AMPK), and acetyl-CoA carboxylase (ACC), enzymes involved in regulating fatty acid oxidation. However, inotropic stimulation selectively increases glucose oxidation in adult hearts. In the neonatal heart, fatty acid oxidation becomes a major energy source, while glucose oxidation remains low. This study tested the hypothesis that increased energy demand imposed by isoproterenol originates from fatty acid oxidation, secondary to increased LKB, AMPK, and ACC phosphorylation. Isolated working hearts from 7-day-old rabbits were perfused with Krebs solution (0.4 mM palmitate, 11 mM glucose, 0.5 mM lactate, and 100 mU/l insulin) with or without isoproterenol (300 nM). Isoproterenol increased myocardial O(2) consumption (in J·g dry wt(-1)·min(-1); 11.0 ± 1.4, n = 8 vs. 7.5 ± 0.8, n = 6, P < 0.05), and the phosphorylation of LKB (in arbitrary density units; 0.87 ± 0.09, n = 6 vs. 0.59 ± 0.08, n = 6, P < 0.05), AMPK (0.82 ± 0.08, n = 6 vs. 0.51 ± 0.06, n = 6, P < 0.05), and ACC-β (1.47 ± 0.14, n = 6 vs. 0.97 ± 0.07, n = 6, P < 0.05), with a concomitant decrease in malonyl-CoA levels (in nmol/g dry wt; 0.9 ± 0.9, n = 8 vs. 7.5 ± 1.3, n = 8, P < 0.05) and increase in palmitate oxidation (in nmol·g dry wt(-1)·min(-1); 272 ± 45, n = 8 vs. 114 ± 9, n = 6, P < 0.05). Glucose and lactate oxidation were increased (in nmol·g dry wt(-1)·min(-1); 253 ± 75, n = 8 vs. 63 ± 15, n = 9, P < 0.05 and 246 ± 43, n = 8 vs. 82 ± 11, n = 6, P < 0.05, respectively), independent of alterations in pyruvate dehydrogenase phosphorylation, but occurred secondary to a decrease in acetyl-CoA content and acetyl-CoA-to-free CoA ratio. As acetyl-CoA levels decrease in response to isoproterenol, despite an acceleration of the rates of palmitate and carbohydrate oxidation, these data suggest net rates of acetyl-CoA utilization exceed the net rates of acetyl-CoA generation.