Successive layer properties and peptide insertion in an assembly of supported mobile phospholipid bilayers on polyion/alkylthiol layer pairs were investigated in a combined optical, electrochemical, and surface topography study using surface plasmon resonance (SPR), cyclic voltarnmetry (CV), and atomic force microscopy (AFM). The use of a long-chain alkylthiol in this assembly was too insulating, and thus, a short-chain alkylthiol was used to probe membrane electrochemical properties. Using AFM, we found that the insulating properties of long-chain and short-chain alkylthiol layers are associated with a continuous layer and domain formation, respectively. Increased insulating properties of the supported bilayers were observed when mixtures of negatively charged and zwitterionic/neutral lipids were used. By attempting to obtain high-resolution images and make depressions in the surface using AFM, we found that this bilayer was more mobile than a bilayer composed completely of negatively charged lipid. A pore-forming antimicrobial and antiviral peptide from porcine leukocytes, protegrin-1, increased the charge transfer through the supported biomembranes. The peptide's influence on the electrochemical and topological properties of the membrane depended on the lipid compositions, although comparable amounts of the peptide were associated with the various membranes. The multilayer surface morphology is quantitatively characterized by using roughness measurements for a large set of data involving root-mean-square roughness (RMS) and power spectra density analysis (PSD). The RMS values obtained for each deposited layer reveal that the surface roughness is characterized in the nanometer and subnanometer range. Surface roughness decreased with each deposited layer in the supported bilayer system but increased with peptide adsorption to the lipid bilayer. A decreased degree of association between the lipid membrane and a mutant protegrin further demonstrates the model membrane as a sensitive tool for studying the mechanisms of antimicrobial peptides.