The capability of microfluidic networks to pattern substrates with proteins is extended to create density gradients of proteins on surfaces. The networks are micromachined in Si, and the substrates are hydrophobic poly(dimethylsiloxane) (PDMS) elastomers. The gradients result from the progressive depletion of proteins in the fluids due to their adsorption onto the PDMS substrate as the solution travels along the microchannel. Forming gradients of rhodamine-tagged antigens on PDMS and binding the antigens with fluorescein-tagged antibodies from solution enable us to study the binding behavior of these partners on a surface: Detection of the fluorescence associated with either partner suggests that recognition of the surface-immobilizcd antigens by an antibody from solution is more effective for a low density of antigens on the surface.