The fatty acid translocase (FAT)/CD36 plays an important role in the acute regulation of fatty acid uptake in muscle tissue. We studied the subcellular distribution of FAT/CD36 in rat cardiac muscle after in vivo insulin stimulation by membrane fractionation and immunoisolation of GLUT4- and FAT/CD36-vesicles. FAT/CD36 was equally present in both plasma and microsomal membranes with no effect of insulin on the cellular distribution, whereas GLUT4 increased 2- to 3-fold in the plasma membrane. FAT/CD36 resides in one intracellular pool, whereas GLUT4 is present in two distinct pools. Immunoadsorption of GLUT4-vesicles indicated that FAT/CD36 is undetectable in these vesicles. Likewise, no GLUT4 could be detected in FAT/CD36-vesicles. These vesicles contain a high amount of Rab11 that remained unaffected after insulin stimulation, whereas Rab11 increased about 3-fold in the GLUT4-vesicles in response to insulin. These data show that GLUT4 and FAT/CD36 do not co-localize in cardiac muscle and that FAT/CD36 is not redistributed in response to insulin in the heart. Rab11 may be involved in endosomal recycling of FAT/CD36, however, insulin-associated Rab11 functions appear to be limited to GLUT4-vesicles. (C) 2002 Elsevier Science (USA). All rights reserved.