The diphtheria toxin (DT) membrane topology was investigated by proteolysis experiments. Diphtheria toxin was incubated with asolectin liposomes at pH5 in order to promote its membrane insertion, and the protein domains located outside the lipid vesicles were digested with proteinase K (which is a non-specific protease). The protected peptides were separated by electrophoresis and identified by microsequence analysis. Their orientation with respect to the lipid bilayer and their accessibility to the aqueous phase were determined by attenuated total reflection Fourier-transform infrared spectroscopy (ATR-FTIR). These data, combined with those provided by proteolytic cleavage with a specific protease (endoproteinase Glu-C), led us to propose a topological model of the N-terminal part of the diphtheria toxin B fragment inserted into the lipid membrane. In this model, two alpha-helices adopt a transmembrane orientation, with their axes parallel to the lipid acyl chains, while a third alpha-helix could adopt a transmembrane topology only in a small proportion of DT molecules.