We present the first results relative to the diffusion process of proteins (human serum albumin) adsorbed on or embedded in poly(sodium 4-styrenesulfonate)/poly(allylamine hydrochloride) polyelectrolyte multilayer films by using fluorescence recovery after photobleaching. At least two populations of adsorbed proteins exist on the top or within the multilayer architectures. One population, which typically represents 50-70% of the adsorbed proteins, corresponds to proteins that are able to diffuse laterally along the surface or in the multilayers, whereas the others diffuse more slowly or seem almost immobile over the experimental time scale. This constitutes a key result for the practical applications of the polyelectrolyte multilayers as targeted biomaterial coatings. Surprisingly, the diffusion coefficients of the mobile proteins that are embedded in the multilayers are comparable with the diffusion coefficients of the mobile proteins adsorbed on top of the multilayers. The diffusion coefficients, which are 1 order of magnitude smaller than on rigid substrates such as silica or poly(methyl methacrylate), are of the order of 10(-10) and 10(-11) cm(2)/s.