Controlling the spatial organization of biomolecules on solid supports with high resolution is important for a wide range of scientific and technological problems. Here we report a study of electron beam lithography (EBL) patterning of a self-assembled monolayer (SAM) of the amide-containing poly(ethylene glycol) (PEG) thiol CH3O(CH2CH2O)(17)-NHCO(CH2)(2)SH on Au and demonstrate the patterning of biomolecular features with dimensions approaching 40 nm. The electron dose dependence of feature size and pattern resolution is studied in detail by atomic force microscopy (AFM), which reveals two distinct patterning mechanisms. At low doses, the pattern formation occurs by SAM ablation in a self-developing process where the feature size is directly dose-dependent. At higher doses, electron beam-induced deposition of material, so-called contamination writing, is seen in the ablated areas of the SAM. The balance between these two mechanisms is shown to depend on the geometry of the pattern. The patterned SAMs were backfilled with fluorescent 40-nm spheres coated with NeutrAvidin. These protein- coated spheres adhered to exposed areas in the SAM with high selectivity. This direct writing approach for patterning bioactive surfaces is a fast and efficient way to produce patterns with a resolution approaching that of single proteins.