The effects of long-chain (LC) fatty acids on rate of heat production (heat rate) and mitochondrial membrane potential () of intact guinea pig cardiac muscle were investigated at 37°C. Heat rate of ventricular trabeculae was measured with microcalorimetry, and was monitored in isolated ventricular myocytes with either JC-1 or tetramethylrhodamine ethyl ester (TMRE). Methyl--cyclodextrin was used as fatty acid carrier. Application of 400 µM oleate or linoleate increased resting heat rate by ~30% and ~25%, respectively. When LC fatty acid was supplied as sole metabolic substrate, resting heat rate was decreased by 3-mercaptopropionic acid. In TMRE-loaded myocytes, neither 40-80 µM oleate nor 40 µM linoleate affected . At a higher concentration (400 µM) both oleate and linoleate increased TMRE fluorescence by ~20% of maximum, obtained using 2,4-dinitrophenol (100 µM), indicating a depolarization of the inner mitochondrial membrane. We conclude that LC fatty acids, at sufficiently high concentration, increase heat rate and decrease in intact cardiac muscle, consistent with a protonophoric uncoupling action. These effects may contribute to the high metabolic rate after reperfusion of postischemic myocardium.