Nanotechnology using enzyme technology to modify surfaces on the nanometer scale and nanostructuring surfaces is an evolving research field. One of the advantages with enzyme assisted nanolithography is the high substrate specificity. Scanning probe microscopy (SPM) in liquid or ambient conditions is highly suited for such nanolithography: we are in particular using atomic force microscopy (AFM) to develop negative nanolithography based on an enzyme and its substrate. The possibility of high accuracy positioning of the SPM probe tip is combined with the activity of an enzyme. There are very few related such studies by other groups [1-3]. In our approach, the enzyme is α-amylase and the substrate starch, whose hydrolysis into sugars by α-amylase is well studied on the macro-scale. The system α-amylase-starch is exploited as a model to demonstrate the proof of principle that the enzyme can be used with SPM methods to locally degrade starch and ultimately write nanopatterns on starch surfaces. Silicon surfaces are covered with smooth and thin layers of starch, whose roughness, morphology and nanomechanical properties are characterized by AFM methods. In the next step the degradation of these starch layers will be demonstrated with an AFM tip functionalized with amylase molecules, either on the apex of a “sharp” AFM tip (outer radius 5-50 nm) or on a micrometer-sized bead of silicon dioxide glued to an AFM cantilever. The α-amylase molecules may be either spontaneously adsorbed or covalently attached to the tip through established functionalization chemistry. Results will be presented related to the structure and quality of the starch layer and its degradation by amylase in solution as observed by AFM and FTIR-spectroscopy and to the activity of amylase immobilized on (silicon/silicon dioxide) surfaces. Preliminary results will be given on the local degradation of starch caused by AFM tips modified by α-amylase and/or by micro droplets of amylase solution released on the substrate by using the AFM tip in a dip-pen configuration. The ultimate goal is to achieve a situation like that illustrated in the drawing above, where a single (or few) α-amylase molecule(s) is attached on the AFM tip so to maintain its enzymatic activity and can be used to write nanopatterns in a layer of starch.