AbstractMale, weanling rats were fed, for up to six weeks, corn oil (CO), rapeseed oil (RSO), partially hydrogenated fat (HF), or a mixture of partially hydrogenated fat and corn oil (HF+CO). The respiratory activity of their isolated heart mitochondria, their hormone‐sensitive lipase activity, and the fatty acid compositions of the phospholipids of the mitochondria were determined. The results indicated that heart mitochondria isolated from rats which had been fed corn oil (CO) had a higher rate of oxygen uptake, showed higher respiratory control ratios, higher ADP/0 ratios and a higher rate of ATP synthesis than the heart mitochondria isolated from those fed rapeseed oil or hydrogenated fats. The oxygen uptake rates of the rat heart mitochondria isolated from each dietary group of rats was in order: oleyl carnitine ≫ erucyl carnitine > elaidyl carnitine. The decreased capacity to oxidize substrate by heart mitochondria which had been isolated from the hearts of rats fed rapeseed or hydrogenated soybean oil as compared with those fed corn oil as a sole source of dietary fat seemed related to the mitochondria lipid composition. The type of dietary fat fed had a pronounced influence on the mitochondrial fatty acid compositions of phosphatidylcholine, phosphatidylethanolamine, and cardiolipin. The lipase activity of the RSO‐fed group did not show any increment with either epinephrine or supplemental ATP treatment. The substrate preference for lipase activity in myocardium was corn oil‐triglycerides > trierucin > trielaidin > tripalmitin. However, cardiac lipid accumulation did not seem related to lipase activity in the myocardium.