Surface composition gradients of extracellular matrix proteins show great promise as tools in understanding cellular adhesion and motility. The formation of a surface coverage gradient of the protein fibronectin (FN) is described. FN is covalently bound to a counterpropagating two-component gradient formed from thiols with carboxylic acid and hydroxyl terminal functional groups using carbodiimide coupling chemistry. Areas of the surface with initial -COOH termination react with amine groups on the FN, while protein adsorption is resisted on the -OH terminated regions. The -COOH/-OH gradient is generated by applying a linear potential profile to a thin Au gold film on which an 11-mercaptoundecanol (MUD) monolayer is present. A gradient in surface coverage of MUD results, which is then backfilled with 11-mercaptoundecanoic acid (MUA). The -COOH/-OH gradient is characterized by tagging with 200 nm diameter, fluorescently doped, amine-modified nanospheres using the same carbodiimide coupling chemistry and imaging with fluorescence microscopy. The surface coverage of FN on MUA determined by surface plasmon resonance reflectometry is 1.08 ng/mm(2), with 0.08 ng/mm(2) of physisorbed protein present on MUD. FN gradients are mapped with FTIR external reflection spectroscopy and imaged with fluorescence microscopy by tagging with anti-FN and FITC-IgG. The gradients exhibit a sigmoid shape, with the width of the gradient region occupying similar to10% of the total film length. When bovine serum albumin is used to block nonspecific cellular adhesion, the adhesion of the 3T3 fibroblasts to these gradients follows the spatial composition profile of FN.