Structural details of the coupling of bacterial surface (S)-layers to the phospholipid, dipalmitoylphosphatidylethanolamine (DPPE), have been characterized using X-ray and neutron reflectometry. We studied the binding and recrystallization of S-protein isolated from B. sphaericus CCM2177 at DPPE monolayers on aqueous surfaces, Particular emphasis has been put on investigations of the lipid/protein interface in a joint refinement of X-ray and neutron data which reveals alterations of the molecular-level organization of the lipid headgroups upon protein binding and recrystallization: Peptide material interpenetrates the phospholipid headgroups almost in its entire depth but does not affect the hydrophobic lipid acyl chains. Consistent with FTIR results, we find that the headgroup hydration is reduced by similar to40% upon peptide interpenetration. On average, the equivalent of similar to65 electrons associated with the peptide, i.e., less than one peptide side group. interacts directly with one DPPE headgroup within the surface film. This suggests that the protein attaches to specific molecular moieties within the lipid monolayer which may form a lateral pattern within the film area that reflects the properties of the monomolecular protein crystal sheet.