Relatively little is known concerning the regulation of uncoupling proteins (UCPs) in the heart. We investigated in the adult rodent heart 1) whether changes in workload, substrate supply, or cytokine (TNF-α) administration affect UCP-2 and UCP-3 expression, and 2) whether peroxisome proliferator-activated receptor α (PPARα) regulates the expression of either UCP-2 or UCP-3. Direct comparisons were made between cardiac and skeletal muscle. UCP-2, UCP-3, and PPARα expression were reduced when cardiac workload was either increased (pressure overload by aortic constriction) or decreased (mechanical unloading by heterotopic transplantation). Similar results were observed during cytokine administration. Reduced dietary fatty acid availability resulted in decreased expression of both cardiac UCP-2 and UCP-3. However, when fatty acid (the natural ligand for PPARα) supply was increased (high-fat feeding, fasting, and STZ-induced diabetes), cardiac UCP-3 but not UCP-2 expression increased. Comparable results were observed in rats treated with the specific PPARα agonist WY-14,643. The level of cardiac UCP-3 but not UCP-2 expression was severely reduced (20-fold) in PPARα-/- mice compared to wild-type mice. These results suggest that in the adult rodent heart, UCP-3 expression is regulated by PPARα. In contrast, cardiac UCP-2 expression is regulated in part by a fatty acid-dependent, PPARα-independent mechanism.