We used infrared attenuated total reflection spectroscopy to study the structure of diphtheria toxin (DT) and its fragments A, B, CB1, and CB4 as a function of the pH in the absence and in the presence of phospholipid vesicles. Binding of DT to asolectin or DL-alpha-dipalmitoylphosphatidylcholine-DL-alpha-dipalmitoylphosphatidic acid liposomes at pH 7.3 results in a 10% increase in its alpha-helix content. At pH 4, in the presence of liposomes, the secondary structure of DT is characterized by the appearance of a beta-sheet structure with strengthened hydrogen bonds which did not exist before pH lowering. DT fragment B displays little conformational change upon pH lowering in the presence of liposomes. However, the alpha-helix content of CB1 increases by 10%, and polarization measurements indicate that the alpha-helices of CB1 at pH 4 are oriented parallel to the lipid acyl chains. On the other hand, the alpha-helix content of CB4 decreases dramatically while the low frequency beta-sheet content increases. Dichroism measurements demonstrate that this sheet lies close to a parallel to the bilayer surface. The fragment A of DT experiences a large conformational change upon pH lowering and binds to the liposome membrane even in the absence of DT fragment B. The conformational modification of DT fragment A is fully reversed when pH is brought back to 7.3.