The membrane-interacting domain that precedes the transmembrane anchor of Ebola glycoprotein has been characterized. This aromatic-rich region is predicted to bind the membrane interface adopting an alpha-helical structure. Peptides representing either the Ebola glycoprotein pre-transmembrane sequence, or a 'scrambled' control with a different hydrophobic-at-interface moment, have been studied. Insertion into lipid monolayers, changes in intrinsic fluorescence and in infrared spectra demonstrated that only the wild-type peptide bound the interface under equilibrium conditions and adopted an alpha-helical conformation. The presence of the raft-associated lipid sphingomyelin did not affect membrane insertion, but it stimulated highly the membrane-destabilizing capacity of the pre-transmembrane sequence. A parallel study of the effects of the viral sequence and of melittin suggests that Ebola glycoprotein pre-transmembrane sequence might target membranes inherently prone to destabilization by lytic peptides.