The association of neuropeptide Y (NPY) with air-water interfaces and with phospholipid monolayers on water subphases and on physiological buffer has been investigated. Surface pressure (pi) versus molecular area (A) relations of the peptide at water surfaces depend on the concentration of the spreading solutions. Independent of that concentration, they show a transition from a low-density state to a high-density state at pi approximately 12 mN/m. Similar features are observed in the NPY adsorption to preformed monolayers (Deltapi(t --> infinity) as a function of pii = pi (t = 0) where t = 0 signifies the time of peptide injection). The transition is also observed in cospread lipid-NPY monolayers and is interpreted as the exclusion of the peptide from the surface layer. The reproducibility of the isotherms after expansion suggests that cospread lipid-peptide monolayers are thermodynamically stable and that the peptide remains associated with the monolayer after exclusion from the lipid surface. A comparison of NPY association with zwitterionic and with anionic lipids as well as a comparison of the interactions on pure water and on physiological buffer suggest that electrostatic attraction plays a major role in the energetics of peptide binding to the membrane surface. Dual label fluorescence microscopy demonstrates that the peptide associates preferentially with the disordered, liquid condensed monolayer phase and also suggests that it self-aggregates upon exceeding a critical surface concentration. A NPY variant with a distorted alpha-helix interacts with the surface as strongly as the natural NPY but expands the monolayers more. This suggests that the helix motif in the peptide is more important for the interaction with the receptor than for binding of the peptide to the membrane surface. In context, these observations attribute a specific role to the membrane in funneling the signal peptide to its membrane receptor.