This Letter describes a new method for production of nanometer-sized protein patterns with precise control of the size and geometry. Self-assembled monolayers (SAMs) are first patterned using nanografting, an atomic force microscopy (AFM)-based lithographic method. Methyl-terminated n-alkanethiols serve as matrix layers within which nanopatterns of thiols with carboxylate or aldehyde terminal groups are fabricated. By controlling solution conditions, we selectively immobilize proteins on the patterned areas, through either electrostatic interactions or covalent binding. In our approach, AFM tips are used for both fabrication and characterization. AFM allows patterned SAMs and proteins to be visualized in situ. The individual proteins within the nanopatterns and their orientations can be clearly resolved from the AFM images.