We have used a novel polyelectrolyte multilayer (PEM) coating consisting of the polyelectrolytes collagen and hyaluronic acid. The build-up by the layer-by-layer deposition technique is outlined by ex situ and in situ ellipsometric measurements. When collagen was added the thickness of the PEM was increased and the refractive index was decreased. Corresponding but opposite effects were noted when hyaluronic acid was added. These changes are considered to be explained by a diffusion mechanism. It was also found that the PEM layers were unstable at physiological pH. However, by crosslinking using N-(3-Di-methylaminopropyl)-N0-ethylcarbodiimide together with N-hydroxysuccinimide a stable PEM layer resulted. These tissue friendly PEM layers are expected to have a great impact in the design of artificial extra cellular matrices. Also, the insertion of fluorescence labels demonstrates the potential for incorporation of other functionalities