Following birth a dramatic increase in fatty acid oxidation occurs in the heart, which has been attributed to an increase in L-carnitine levels and a switch from the liver (L) to muscle (M) isoform of carnitine palmitoyltransferase (CPT) 1. However, since M-CPT 1 has a lower affinity for L-carnitine, and is more sensitive to inhibition by malonyl CoA, a potent endogenous regulator of fatty acid oxidation in the heart, a switch from the L-CPT 1 isoform to the M-CPT 1 isoform should theoretically result in a decrease in fatty acid oxidation. Due to this discrepancy we assessed the importance of changes in myocardial L-carnitine content, CPT 1 isoform expression, and CPT 1 kinetics to the maturation of fatty acid oxidation that occurs in the newborn rabbit heart. Although fatty acid oxidation rates increased dramatically between 1 and 14 days following birth, myocardial L-carnitine concentrations did not show an age dependent increase (cellular concentrations ranged from 2.0 mM to 1.4 mM). Expression of L-CPT mRNA increased immediately after birth, but returned to fetal levels by 10 days of age. In contrast, M-CPT 1 mRNA expression levels did not show a change between 1-day and 7-days following birth (a time frame in which fatty acid oxidation dramatically increases), but then increased by 3 fold and 30 fold by 10 days and 14 days of age, respectively. Despite these changes in CPT 1 expression, the Km for carnitine did not change between 1-day and 10-days following birth. The IC₅₀ for malonyl CoA inhibition of CPT 1 was also similar between 1-day and 10-day old hearts. However, malonyl CoA levels dramatically decreased following birth, which was accompanied by a decrease in acetyl CoA carboxylase activity (which synthesizes malonyl CoA) and a significant increase in the activity of malonyl CoA decarboxylase (which degrades malonyl CoA). Although fatty acid oxidation rates increase following birth, glucose oxidation rates remained low. This occurred even though the activity of pyruvate dehydrogenase, the rate-limiting enzyme for fatty acid oxidation, and the maximal capacity for glucose oxidation significantly increased. Combined, these data suggest changes in L-carnitine content and CPT 1 isoform expression and kinetics are not the major reason for the increase in fatty acid oxidation following birth. Instead, our data suggest that a decrease in malonyl CoA control of CPT 1 is primarily responsible for the increase in fatty acid oxidation and the low glucose oxidation rates seen in the newborn heart.