This study investigates the role of lipid rafts and caveolae, a subclass of lipid raft microdomains, in the binding and uptake of long-chain fatty acids (LCFA) by 3T3-L1 cells during differentiation. Disruption of lipid rafts by β-cyclodextrin (βCD) or selective inhibition of caveolae by overexpression of a dominant-negative mutant of caveolin-3 (Cav^DGV) resulted in disassembly of caveolae structures at the cell surface, as assessed by electron microscopy. While in 3T3-L1 fibroblasts, which express few caveolae, Cav^DGV or βCD had no effect on LCFA uptake, in 3T3-L1 adipocytes the same treatments decreased the level of [³H]oleic acid uptake by up to 55 ± 8 and 49 ± 7%, respectively. In contrast, cholesterol loading of 3T3-L1 adipocytes resulted in a 4-fold increase in the extent of caveolin-1 expression and a 1.7-fold increase in the level of LCFA uptake. Both the inhibitory and enhancing effects of these treatments were constantly increasing with the [³H]oleic acid incubation time up to 5 min. Incubation of 3T3-L1 adipocytes with [³H]stearate followed by isolation of a caveolin-1 positive detergent-resistant membrane (DRM) fraction revealed that [³H]stearate binds to caveolae. Fatty acid translocase (FAT/CD36) was found to be present in this DRM fraction as well. Our data thus strongly indicate a critical involvement of lipid rafts in the binding and uptake of LCFA into 3T3-L1 adipocytes. Furthermore, our findings suggest that caveolae play a pivotal role in lipid raft-dependent LCFA uptake. This transport mechanism is induced in conjunction with cell differentiation and might be mediated by FAT/CD36.