Peroxisome proliferator-activated receptor α (PPARα) is a transcriptional regulator of the expression of mitochondrial thioesterase I (MTE-I) and uncoupling protein 3 (UCP3), which are induced in the heart at the mRNA level in response to diabetes. Little is known about the regulation of protein expression of MTE-I and UCP3 or about MTE-I activity; thus, we investigated the effects of diabetes and treatment with a PPARα agonist on these parameters. Rats were either made diabetic with streptozotocin (55 mg/kg ip) and maintained for 10-14 days or treated with the PPARα agonist fenofibrate (300 mg/kg/day) for 4 weeks. MTE-I and UCP3 protein expression, MTE-1 activity, palmitate export, and oxidative phosphorylation were measured in isolated cardiac mitochondria. Diabetes and fenofibrate increased cardiac MTE-I mRNA, protein, and activity (∼4-fold compared with controls). This increase in activity was matched by a 6-fold increase in palmitate export in fenofibrate-treated animals, despite there being no effect in either group on UCP3 protein expression. Both diabetes and fenofibrate caused significant decreases in state III respiration of isolated mitochondria with pyruvate + malate as the substrate, but only diabetes reduced state III rates with palmitoylcarnitine. Both diabetes and specific PPARα activation increased MTE-I protein, activity, and palmitate export in the heart, with little effect on UCP3 protein expression. Copyright © 2007 by the American Society for Biochemistry and Molecular Biology, Inc.