Proteolytic activation, oligomerization, and membrane insertion are three steps that precede channel formation by the bacterial toxin aerolysin. Using attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) and hydrogen-deuterium exchange, the structural changes associated with each step were analyzed. Our results show that activation induces a significant change in secondary structure, characterized by a decrease in random structure and an increase in beta-sheet content. We show that release of the propeptide is essential for this conformational change to occur and that changes are not restricted to the vicinity of the cleavage site but appear to propagate along the molecule. In contrast, subsequent oligomerization of the mature toxin does not involve any change in overall secondary structure but does involve a modification of the tertiary interactions. Finally, insertion of the heptameric complex into dimyristoylphosphatidylcholine vesicles also occurs without major modification of the secondary structure. Studies on the orientations of the secondary structures of the heptamer in the lipid bilayer have also been performed.