The lipid composition and the curvature of biological membranes are limiting steps for peptide interactions with living cells and liposomes. Cholesterol has been proven to be essential for filovirus replication, and the entry of the Ebola and Marburg viruses is inhibited after cholesterol depletion of the target cells. In cells not depleted of cholesterol, viral proteins co-localize with caveolin after internalization. Caveolae are vesicles enriched with cholesterol and sphingolipids and have been shown to be involved in a wide range of biological events such as cellular entry by certain viruses. In this work, we depleted cholesterol from cells to understand its importance in the mechanism of membrane fusion, an early step in the Ebola infection cycle. Since Vero and BHK-21 mammalian cells are permissive to infection mediated by the Ebola virus, initial attempts were performed by using those cells. b-cyclodextrins were used to since they are very effective to selectively extract cholesterol from membranes of intact cells without binding or insertion into the plasma membrane. Vero and BHK-21 cells were treated with increasing concentrations of MbCD for 30 min at 37uC and then assayed for cholesterol quantification. As shown in Fig. 1A, cholesterol depletion was dose-dependent for Vero and BHK-21 cells. In addition, insect cells, previously grown in medium with cholesterol, were assayed as a cellular control of low cholesterol content cells. Insect cells are cholesterol auxotrophs and can be depleted of cholesterol by growth in delipidated serum. As observed in Fig. 1A, the cholesterol content of C6/36 cells was maintained after incubation with up to 12 mM MbCD. However, upon incubation with 20 and 24 mM MbCD, it was not possible to detect cholesterol due to the low cell adhesion induced by depletion. To determine the effect of MbCD on cell viability, Vero, BHK-21 and C6/36 cells were incubated in the absence or in the presence of MbCD. At the same time we added MTT reagents to prevent cellular loss during the washing step. In general, insect and mammalian cell monolayers were intact after 30 min incubation with up to 16 or 24 mM of MbCD, respectively. Fig. 1B shows that insect cells were more affected by cholesterol depletion than mammalian cells. Indeed, 16 mM MbCD was able to decrease 50–60% of cholesterol in mammalian and insect cells but only affected the viability of insect cells. Thus, our results showed that some different MbCD concentrations can induce similar levels of cholesterol depletion but different responses in cellular viability. In our studies, low MbCD concentrations, which cause depletion of cholesterol but do not affect the cellular viability, were chosen to examine the role played by cholesterol during protein-membrane interaction. Previous studies had shown that low BYL719 endosomal pH is required for infection and cell-cell fusion mediated by Ebola virus GP and that low pH is required for optimal functioning of cathepsin B and L.