The ability to pattern a surface with proteins on both the nanometer and the micrometer scale has attracted considerable interest due to its applications in the fields of biomaterials, biosensors, and cell adhesion. Here, we describe a simple particle lithography technique to fabricate substrates with hexagonally patterned dots of protein surrounded by a protein-repellent layer of poly(ethylene glycol). Using this bottom-up approach, dot arrays of three different proteins (fibrinogen, P-selectin, and human serum albumin) were fabricated. The size of the protein dots (450 nm to 1.1 microm) was independent of the protein immobilized but could be varied by changing the size of the latex spheres (diameter=2-10 microm) utilized in assembling the lithographic bead monolayer. These results suggest that this technique can be extended to other biomolecules and will be useful in applications where arrays of protein dots are desired.