In addition to providing morphological support, the actin cytoskeleton is involved in cell motility, endocytosis and intracellular transport. Cytoskeletal interaction with the membrane is an essential part of these processes and is mediated by accesory proteins. The ERM proteins ezrin, radixin and moesin are known to act as linker proteins, connecting the actin cytoskeleton to membrane components such as phosphatidylinositol 4,5-bisphosphate and CD44. The different ERM proteins probably share overlapping functions, but are expressed in a tissue and developmental specific manner. In cultured HeLa cells, mainly ezrin and moesin are reported to be present. To study the role of the ERM proteins in endocytosis and intracellular transport, we treated the cells with siRNA against ezrin and moesin. We then investigated the effect of the knockdown on the binding and retrograde transport of the Shiga toxin (Stx) from the cell exterior, via endosomes, the Golgi apparatus and the endoplasmic reticulum to the cytosol. By siRNA knockdown of ezrin and/or moesin in these cells, we show a decrease in the binding of Stx to the cell surface as well as a decrease in the level of the Stx receptor Gb3 at the plasma membrane. We also show an even larger decrease in the amount of Stx transported from the plasma membrane to the Golgi apparatus and the ER.