Lipid rafts are proposed to be ordered domains within the plasma membrane that comprise sphingolipids and sterols. They control numerous cellular processes, such as membrane lipid sorting and signalling pathways. For example, the removal of sphingomyelin or sterol affects endo- or phagocytotic trafficking, which suggests a role for lipid rafts in these processes. By substituting various sterols for cholesterol instead of removing it, Deborah Brown, Erwin London and colleagues (p. 2682) now assess the role of sterols and ordered plasma membrane domains during endocytosis. The authors find that a group of sterols is capable to reversibly inhibit both clathrin-mediated and clathrin-independent endocytosis. Interestingly, sterols need to be able to promote lipid raft formation for endocytosis to occur; however, this is not sufficient, as one sterol lacking the 3β-hydroxyl headgroup supported lipid raft formation, but not endocytosis. The authors further show that the presence of a double-bond in the sterol B-ring or the presence of an aliphatic tail identical to the structure of cholesterol are neither necessary nor sufficient for endocytosis. This study shows that the structure of sterol in the plasma membrane is important for both clathrin-mediated and -independent endocytosis, and that the ability to form ordered membrane domains and the presence of a 3β-OH group are essential for sterols to perform this task.