Controlling the organization of proteins on surfaces provides a powerful biochemical tool for determining how cells interpret the spatial distribution of local signaling molecules. Here, we describe a general high fidelity approach based on electron beam writing to pattern the functional properties of protein-coated surfaces at length scales ranging from tens of nanometers to millimeters. A silicon substrate is first coated with the extracellular matrix protein fibronectin, which is then locally inactivated by exposure to a highly focused electron beam. Biochemical inactivation of the protein is established by the loss of antibody binding to the fibronectin. Functional inactivation is determined by the inability of cells to spread or form focal adhesions on the inactivated substrate, resulting in cell shapes constrained to the pattern, while they do both (and are unconstrained) on the remaining fibronectin. These protein patterns have very high fidelity, and typical patterns agree with the input dimensions of the pattern to within 2%. Further, the feature edges are well defined and approach molecular dimensions in roughness. Inactivation is shown to be dose dependent with observable suppression of the specific binding at 2 mu C cm(-2) and complete removal of biochemical activity at similar to 50 mu C cm(-2) for 5 keV electrons. The critical dose for inactivation also depends on accelerating voltage, and complete loss of antibody binding was achieved at similar to 4-7 mu C cm(-2) for 1 keV electrons, which corresponds to similar to 50-90 electrons per cross-sectional area of a whole fibronectin dimer and similar to 2-4 electrons per type III fibronectin domain. AFM analysis of the pattern surfaces revealed that electron beam exposure does not remove appreciable amounts of material from the surface, suggesting that the patterning mechanism involves local inactivation rather than the ablation that has been observed in several organic thin film systems.