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In this work, the specific and nonspecific binding of proteins to planar surfaces was investigated. The surfaces were first modified by the spontaneous adsorption of a number of synthesized 1,2-diacyl-sn-glycero-3-phosphatidylcholine lipids with different ω-mercapto fatty acids. The influence of packing density, order, fluidity, and mobility of the head groups of these supported thiolipid monolayers on the nonspecific adsorption of proteins was systematically investigated. Nonspecific binding of fibrinogen, IgG, BSA, and rabbit serum to such surfaces was monitored on-line by surface plasmon resonance spectroscopy (SPR) and was found to be exceptionally low. Characterization of the supported thiolipid monolayers by grazing incidence infrared spectroscopy, wetting, impedance spectroscopy, calorimetry, and SPR indicates that protein adsorption is influenced by the nature of the fatty acids and suggests that besides the molecular structure of the layer-forming molecules, other supramolecular aspects such as flexibility of the immobilized lipid layer or protrusion of the lipid head groups play a role. These investigations point the way for the construction of even more highly protein repellent surfaces with potential technical applications in the medical (implants) and diagnostic fields (biosensors). To investigate specific protein-surface interactions, acetylcholine containing ligand molecules of the general molecular structure HS(CH2)15(CO)O(CH2CH2O)nCH2(CO)OCH2CH2N+(CH3)3 X- (n = 4, 6, 13) were synthesized. They allow acetylcholine, the natural agonist of the acetylcholine receptor, to be covalently immobilized on a surface. Mixed layers of thiolipids and ligand molecules were formed by self-assembly on planar gold substrates and specific interactions between the immobilized acetylcholine and the nicotinic acetylcholine receptor (nAchR) from Torpedo Californica have were investigated on-line by SPR. Though it seems that the receptor binds specifically, bound receptor could be only partially displaced from the surface by excess free ligand. However, further improvement of the sensor surfaces will surely provide in the future a powerful tool for the investigation of membrane proteins and transmembrane communication.