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 have been investigated at 37 °C. Heat rate of ventricular trabeculae was measured using microcalorimetry and was monitored in isolated ventricular myocytes using either JC-1 or TMRE. Methyl-ß-cyclodextrin was used as fatty acid carrier. Application of 400 µM oleate or linoleate increased resting heat rate by, respectively, ~30 % and ~25 %. 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 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 following reperfusion of post-ischemic myocardium.