A novel technique for the quantitative observation of cell migration along linear gradient substratesfunctionalized with adhesive proteins is presented. Gradients of the cell adhesion molecule fibronectin aregenerated by the cross diffusion of functionalizable alkanethiols on gold and characterized by X-rayphotoelectron spectroscopy and surface plasmon resonance. Two distinct migration assays are describedthat characterize the movement of either sparsely populated noncontacting cells or a confluent monolayerof cells into free space. The drift speed of bovine aortic endothelial cells is measured and shown to increasealong a fibronectin gradient when compared to a uniform control substrate using both assays. The resultsof these experiments establish reproducible conditions for studies of cell migration on gradients of surface-bound ligands.