A method has been developed to spatially define the immobilization of proteins on surfaces using the classic avidin-biotin link, for which a wide variety of biochemical reagents are commercially available. A derivative of biotin bearing a photoremovable nitrobenzyl group (MeNPOC-biotin) has been prepared in a form suitable for simple linkage to biomolecules and surfaces. It has been used to functionalize bovine serum albumin (BSA) to form MeNPOC-biotin-BSA, which can then be coated onto glass. On photolithographic patterning of the surface, biotins are freed in the irradiated areas, permitting avidin to be localized at the irradiated sites. Subsequent addition of a biotinylated molecule permits its site-specific localization. Patterning of a biotinylated antibody and dye-labeled avidins or streptavidin using this reagent has been demonstrated by fluorescence microscopy.