Electrochemical impedance spectroscopy, Fourier transform infrared reflection-absorption spectroscopy, and cyclic voltammetry were employed to characterize polyelectrolyte multilayers (PEMs) fabricated with poly-(styrenesulfonate) as the polyanion and the polypeptides poly-L-histidine, poly-L-lysine, and poly-L-arginine as polycations. The layer-by-layer electrostatic assembly was produced onto alkanethiol-modified gold surfaces. The frequency response reveals that the effect of the number of layers seems to be related to a progressive reduction in the active area of the PEM-modified electrodes. The active area after the deposition of seven layers can be lower than 10% of its original value. The film surface is then inhomogeneous with respect to the transport of the electroactive species and has spots through which transport is quite favored. These structural features of the PEM have been taken into account in the theoretical model of ion transport and very good agreement with the experimental impedance results has been found.