The currently accepted paradigm is that fatty acid (FA) metabolism is attenuated in the hypertrophied and failing heart. This is thought to be due to a decreased expression of genes involved in FA uptake and metabolism, secondary to a decline in peroxisome proliferator-activated receptor α (PPARα) activity. The aim of the present study was to investigate the relation between the expression of FA handling genes and FA oxidation rate during cardiac hypertrophy using neonatal rat cardiomyocytes (NCM) from humanely killed neonatal rats as a model system. Therefore, NCM were exposed to the α1-adrenergic agonist phenylephrine (PE, 10^-5 M) or thyroid hormone (T3, 10^-8 M) in the absence or presence of natural (FA) or synthetic (Wy-14,643) PPARα ligands for 48 h. Experiments were performed at least in triplicate. Statistical analysis of group differences was performed with one-way ANOVA, followed by two-tailed Student’s t test. PE and T3 stimuli induced distinct cellular phenotypes as was evident from cell morphology and the differential expression of the hypertrophic marker gene atrial natriuretic factor and sarcoplasmic reticulum Ca²⁺-ATPase 2a. PE and T3 affected neither basal expression nor PPAR-mediated induction of a panel of lipid metabolising genes at the mRNA or protein level. In transient transfection studies the basal activity of the human muscle-type carnitine palmitoyl transferase-I promoter was not affected by PE or T3. PE, but not T3, markedly increased the oxidation of [¹⁴C]-palmitate from 0.15±0.04 to 0.45±0.16 nmol palmitate mg^-1 min^-1 (p<0.05), whereas the oxidation rates of glucose (0.70±0.09 nmol glucose mg^-1 min^-1) and pyruvate (0.50±0.10 nmol pyruvate mg^-1 min^-1) remained constant. The PPAR-mediated increase in [¹⁴C]-palmitate oxidation rate was not affected by PE or T3. The present findings point to a selective increase of FA oxidation capacity in PE-stimulated hypertrophied cardiomyocytes independent of changes in the expression of FA handling genes, thereby challenging the current paradigm that cardiac hypertrophy is associated with a decline in PPARα activity and, consequently, impaired fatty acid metabolism.