The primary aim of the present study was to investigate in which cellular compartments fatty acid trans-locase CD36 (FAT/CD36) is localized. Intact and fully functional skeletal muscle mitochondria were isolated from lean and obese female Zucker rats and from 10 healthy male individuals. FAT/CD36 could not be detected in the isolated mitochondria, whereas the mitochondrial marker F(1)ATPase-beta was clearly detected using immunoblotting. Lack of markers for other membrane structures indicated that the mitochondria were not contaminated with membranes known to contain FAT/CD36. In addition, fluorescence immunocytochemistry was performed on single muscle fibers dissected from soleus muscle of lean and obese Zucker rats and from the vastus lateralis muscle from humans. Costaining against FAT/CD36 and MitoNEET clearly show that FAT/CD36 is highly present in sarcolemma and it also associates with some vesicle-like intracellular compartments. However, FAT/CD36 protein was not detected in mitochondrial membranes, supporting the biochemical findings. Based on the presented data, FAT/CD36 seems to be abundantly expressed in sarcolemma and in vesicle-like structures throughout the muscle cell. However, FAT/CD36 is not present in mitochondria in rat or human skeletal muscle. Thus, the functional role of FAT/CD36 in lipid transport seems primarily to be allocated to the plasma membrane in skeletal muscle.