Bipolar amphiphiles belonging to a,w-bis(4-amidinophenoxy)alkanes self-assemble in neutral or alkaline aqueous solution on acid-functionalised surfaces to form pH-switchable layers, with the amphiphiles oriented nearly perpendicular to the substrate. Layer thicknesses estimated by in situ ellipsometry, surface plasmon spectroscopy (SPS) or neutron reflection measurements correlated, within a certain amphiphile size range, with the molecular length of the amphiphile. Above a certain chain length, the characterisation by infrared reflection absorption spectroscopy (IRAS) indicated a crystallinelike order of the layered amphiphiles and a tendancy for bilayer formation was observed. These layers were stable and charge selective towards plasma proteins and charged surfactants.The layer stability was strongly pH dependant and no self-assembly was observed in acidic solutions. By cycling the pH between 9 and 2 rapid disassembly and reassembly of the layers was thus observed. The pH-switchable assembly can be used to restore a chemically selective interface after binding of a target molecule, for enhancement of sensor responses and potentially for optoelectronic or molecular electronic applications.