Rationale: Long chain fatty acids (LCFAs) are the preferred substrate for energy provision in hearts. However, the contribution of endogenous triacylglyceride (TAG) turnover to LCFA oxidation and the overall dependence of mitochondrial oxidation on endogenous lipid is largely unstudied. Objective: We sought to determine the role of TAG turnover in supporting LCFA oxidation and the influence of the lipid-activated nuclear receptor, proliferator-activated receptor (PPAR)α, on this balance. Methods and results: Palmitoyl turnover within TAG and palmitate oxidation rates were quantified in isolated hearts, from normal mice (nontransgenic) and mice with cardiac-specific overexpression of PPARα (MHC-PPARα). Turnover of palmitoyl units within TAG, and thus palmitoyl-coenzyme A recycling, in nontransgenic (4.5±2.3 μmol/min per gram dry weight) was 3.75-fold faster than palmitate oxidation (1.2±0.4). This high rate of palmitoyl unit turnover indicates preferential oxidation of palmitoyl units derived from TAG in normal hearts. PPARα overexpression augmented TAG turnover 3-fold over nontransgenic hearts, despite similar fractions of acetyl-coenzyme A synthesis from palmitate and oxygen use at the same workload. Palmitoyl turnover within TAG of MHC-PPARα hearts (16.2±2.9, P<0.05) was 12.5-fold faster than oxidation (1.3±0.2). Elevated TAG turnover in MHC-PPARα correlated with increased mRNA for enzymes involved in both TAG synthesis, Gpam (glycerol-3-phosphate acyltransferase, mitochondrial), Dgat1 (diacylglycerol acetyltransferase 1), and Agpat3 (1-acylglycerol-3-phospate O-acyltransferase 3), and lipolysis, Pnliprp1 (pancreatic lipase related protein 1). Conclusions: The role of endogenous TAG in supporting β-oxidation in the normal heart is much more dynamic than previously thought, and lipolysis provides the bulk of LCFA for oxidation. Accelerated palmitoyl turnover in TAG, attributable to chronic PPARα activation, results in near requisite oxidation of LCFAs from TAG. © 2010 American Heart Association, Inc.