Summary The effect of carnitine on fatty acid synthesis in myocardium has been studied with isolated perfused rat hearts. Circulating carnitine (0.5 mM or 5 mM) depressed acetate (5 mM) incorporation into heart lipids in perfused heart preparations, and this effect was due primarily to reduced incorporation into tissue fatty acids and triglycerides. There was also stimulation of acetate oxidation to 14CO2 by the higher level of perfusing carnitine. Carnitine significantly depressed pyruvate (5 mM) uptake by perfused rat heart but had no effect on oxidation of pyruvate to 14CO2, nor on incorporation of pyruvate into heart lipids. With 105,000g supernatant fraction of heart, fatty acid synthesis from acetate was markedly depressed in the presence of carnitine, while with liver supernatant fraction there was a 40% stimulation of fatty acid synthesis by carnitine. These changes were again reflected primarily in the unesterified fatty acid fraction in each tissue.