It is well known that muscle contraction and insulin can independently translocate GLUT-4 from an intracellular depot to the plasma membrane. Recently, we have shown that the fatty acid transporter FAT/CD36 is translocated from an intracellular depot to the plasma membrane by muscle contraction (<30 min) (Bonen et al. J Biol Chem 275: 14501-14508, 2000). In the present study, we examined whether insulin also induced the translocation of FAT/CD36 in rat skeletal muscle. In studies in perfused rat hindlimb muscles, we observed that insulin increased fatty acid uptake by +51%. Insulin increased the rate of palmitate incorporation into triacylglycerols, diacylglycerols, and phospholipids (P < 0.05) while reducing muscle palmitate oxidation (P < 0.05). Perfusing rat hindlimb muscles with insulin increased plasma membrane FAT/CD36 by +48% (P < 0.05), whereas concomitantly the intracellular FAT/CD36 depot was reduced by 68% (P < 0.05). These insulin-induced effects on FAT/CD36 translocation were inhibited by the phosphatidylinositol 3-kinase inhibitor LY-294002. Thus these studies have shown for the first time that insulin can induce the translocation of FAT/CD36 from an intracellular depot to the plasma membrane. This reveals a previously unknown level of regulation of fatty acid transport by insulin and may well have important consequences in furthering our understanding of the relation between fatty acid metabolism and insulin resistance.