Polyelectrolyte multilayers constructed from polypeptides present secondary structures similar to those found in proteins (R-helices and β-sheets). These secondary structures are used as a tool to investigate the buildup and internal stability of multilayer films by means of Fourier transform infrared spectroscopy. Special attention is focused on the β-sheet contribution to the amide I band. Two main problems are addressed: (i) Does there exist a correlation between the local structure of the polypeptide multilayers and their corresponding polyanion/polycation complexes in solution? (ii) How stable is the local structure of these multilayers toward external stresses such as pH jumps, temperature rise, and changes of the nature of the outer layers of the film? Four different polypeptide multilayers, poly(L-glutamic acid)/poly(Llysine) (PGA/PLL), poly(L-aspartic acid)/poly(L-lysine), poly(L-glutamic acid)/poly(D-lysine), and poly(Lglutamic acid)/poly(L-ornithine), are studied. It is shown that the film secondary structures always closely resemble those of their corresponding complexes in solution. For example, the absence of β-sheet structures in the films correlates with their absence in solution. This shows the strong similarity between the physical processes leading to the formation of polypeptide complexes in solution and those involved in the multilayer formation. The secondary structures of (PGA/PLL)n films appear very stable against pH jumps for pH values ranging between 4 and 10.5. On the other hand, the sudden contact of a film constructed at pH 7.4 with a solution at pH 1.5 or 13.5 leads to a strong reduction of its β-sheet content together with a partial or total dissolution of the film. The structural response of a (PGA/PLL) n film to a temperature rise up to 89 °C depends on the way in which the temperature increase is performed: a slow temperature increase induces a reversible decrease of the β-sheet content at the expense of the R-helices. On the contrary, when the film is heated rapidly, the β-sheet content increases and a further increase is observed during cooling to room temperature. Finally, the deposition of poly(styrene sulfonate)/poly(allylamine) (PSS/PAH) bilayers on top of (PLL/PGA) n films leads to the total disappearance of the β-sheets. This seems to be related to the diffusion of PSS chains into the film during the first PSS deposition steps and an exchange of PGA molecules of the film by PSS ones deep in the architecture. Such an exchange process between two polyelectrolytes of different nature inside a multilayer architecture was, to the authors' knowledge, never observed before.