A method is described for immobilization of receptors, antibodies, or other macromolecules at precise locations on solid substrates. We have combined photolithographic techniques with the use of a "caged" biotin analogue that has been covalently linked to the substrate surface. Exposure to near UV light through a photolithographic mask yields biotin sites for streptavidin binding. Biotinylated macromolecules are then immobilized via a biotin-streptavidin-biotin bridge. Molecules may be attached at selected locations by carrying out repeated rounds of exposure, streptavidin binding, and application of the biotinylated reagent. We have demonstrated the immobilization of fluorescein-streptavidin molecules in 500 mu m x 500 mu m sites, and the localization of two biotinylated antibodies at different sites on a planar substrate surface. We anticipate that the technique will prove useful in drug screening, diagnostics, and biosensor applications.