Ursolic acid (1), a pentacyclic triterpene acid, is one of the major components of certain traditional medicinal plants and possesses a wide range of biological effects, such as anti-inflammatory, antioxidative, and cytotoxic activities. Furthermore, 1, when present at 1.6-5 ng/mL concentrations in commercial herbal preparations used for patients with cardiac disorders, may also exert pro-cardiac activities. There are several indirect suggestions that the cardioprotective mechanism of ursolic acid could involve the mitochondria; however the mechanism of action is still not known. Therefore, the effects of 0.4-200 ng/mL ursolic acid (1) on the functions of isolated rat heart mitochondria oxidizing either pyruvate and malate, succinate, or palmitoyl-l-carnitine plus malate were investigated. It was found that 1 induced a statistically significant uncoupling of oxidative phosphorylation. A statistically significant decrease in H₂O₂ production in the mitochondria was observed after incubation with 5 ng/mL 1. This effect was comparable to the effectiveness of the classical uncoupler carbonyl cyanide 3-chlorophenylhydrazone. Since mild mitochondrial uncoupling has been proposed as one of the mechanisms of cardioprotection, the present results indicate that ursolic acid (1) has potential use as a cardioprotective compound.