A novel method of producing a poly(ethylene glycol) (PEG)-based gradient matrix that varies gradually in thickness from 0 to 500 A over a distance of 5-20 mm is presented. The gradient matrix is graft copolymerized from a mixture of PEG methacrylates onto organic thin films providing free radical polymerization sites initiated by UV irradiation at 254 nm. The films used as grafting platforms consist of either a spin-coated cycloolefin polymer or a self-assembled monolayer on planar gold. The thickness/irradiation gradient is realized by means of a moving shutter that slowly uncovers the modified gold substrate. The structural and functional characteristics of the gradient matrix are investigated with respect to thickness profile, degree of carboxylation, and subsequent immobilization of two model proteins of different sizes and shapes. These characteristics are studied with ellipsometry and infrared reflection-absorption microscopy using a grazing angle objective. It is revealed that the relatively small carboxylation agent used offers homogeneous activation throughout the gradient, even in the thick areas, whereas the diffusion/interpenetration and subsequent immobilization of large proteins is partially hindered. This is crucial information in biosensor design that can be easily obtained from a gradient experiment on a single sample. Moreover, the partially hindered protein interpenetration, the marginal swelling upon hydration, and the unspecific nature of the graft polymerization suggest a matrix growth mechanism that favors the formation of a bushlike polymer structure with a certain degree of cross linking.