Malonyl-CoA can be formed within the mitochondria, peroxisomes, and cytosol of mammalian cells. Besides being an intermediate in the pathways of de novo fatty acid biosynthesis and fatty acid elongation, malonyl-CoA has an important signaling function through its allosteric inhibition of carnitine palmitoyltransferase 1, the enzyme that normally exerts flux control over mitochondrial beta-oxidation. Malonyl-CoA is rapidly turned over in mammalian cells, and the activities of acetyl-CoA carboxylase and malonyl-CoA decarboxylase are important determinants of its cytosolic concentration. It is now recognized that malonyl-CoA participates in a diverse range of physiological or pathological responses and systems. These include the ketogenic response of the liver to fasting and diabetes, carbohydrate versus fat fuel selection in muscle tissues, metabolic changes in muscle during contracture, alterations in fatty acid metabolism during cardiac ischemia and postischemic reperfusion, stimulation of B cell insulin secretion by glucose, and the hypothalamic control of appetite.