Protein multilayers were prepared on silica surfaces by up to 20 alternating incubations of a biotin-protein conjugate and polymerized streptavidin. Spectroscopic ellipsometry (350-750 nm) was used to investigate the physical thickness and the dispersion of the protein layer after each incubation step. Both parameters could be determined independently for films formed by 5 to 15 incubations. A single homogeneous protein layer was assumed for evaluation. This determination of layer parameters was limited by high correlation coefficients for less than 5 incubation steps. Deviations from the homogeneous single layer model were found for more than 15 incubation steps. The growth of the layer was reproducible, with a thickness increase of about 18.75 nm per incubation. An almost constant refractive index n(D) of 1.384 +/- 0.002 was found for the multilayer system. A protein mass deposition of 4.74 ng/mm(2) was calculated per incubation step. The protein concentration of the layer was estimated of about 0.27 g/ml. The affinity system investigated is quite simple and may well serve as a prototype system in the characterization of optical and other transducers for affinity reactions.