In newborn rabbits, fatty acid oxidation rates in the heart significantly increase between 1 and 7 days after birth. This is due in part to a decrease in malonyl coenzyme A (CoA) production by acetyl CoA carboxylase (ACC). In other tissues, 5'-AMP-activated protein kinase (AMPK) can phosphorylate and inhibit ACC activity. In this study, we show that 1- and 7-day-old rabbit hearts have a high AMPK activity, with AMPK expression and activity being greatest in 7-day-old hearts. Hearts were also perfused in the Langendorff mode with Krebs-Henseleit buffer containing 0.4 mmol/L [14C]palmitate and 11 mmol/L glucose +/- 100 microU/mL insulin. In the absence of insulin, fatty acid oxidation rates were significantly higher in 7-day-old hearts compared with 1-day-old hearts. AMPK activity was also greater in 7-day-old hearts compared with 1-day-old hearts (909 +/- 60 and 585 +/- 75 pmol.min-1.mg protein-1, respectively; P < .05). In 1-day-old hearts, the presence of insulin resulted in a significant decrease in AMPK activity, an increase in ACC activity, and a decrease in fatty acid oxidation rates. In 7-day-old hearts, AMPK activity was also decreased by insulin, although ACC activity remained low and fatty acid oxidation rates remained high. Stimulation of AMPK in 7-day-old hearts with 200 mumol/L 5-amino 4-imidazolecarboxamide ribotide resulted in a further decrease in ACC activity and an increase in fatty acid oxidation rates. These data suggest that AMPK, ACC, and fatty acid oxidation are sensitive to insulin in 1-day-old rabbit hearts and that the decrease in circulating insulin levels seen after birth leads to an increased activity of AMPK. This can then lead to a phosphorylation and inhibition of ACC activity, with a resultant increase in fatty acid oxidation rates.