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Poly( l-lysine) (PLL)/hyaluronic acid (HA) multilayers are films whose thickness increases exponentially with the number of deposition steps. Such a growth process was attributed to the diffusion, in and out of the whole film, of at least one of the polyelectrolytes constituting the film. In the case of PLL/HA, PLL is known to be the diffusing species. In order to better understand the growth mechanism of such films, it is of primary importance to well characterize the diffusion process of the polyelectrolytes in the multilayer. This process is studied here by fluorescence recovery after pattern photobleaching. We show that the diffusion behavior is different when we consider either PLL chains that are deposited on top of the film or PLL chains embedded in the film, even below only one HA layer. For chains that are embedded, we find two populations: a mobile one with a diffusion coefficient, D, of the order of 0.1 µm 2·s −1and a population that appears immobile ( D< 0.001 µm 2·s −1). For chains deposited on top of the multilayer, a third population appears which is rapidly diffusing ( D≅ 1 µm 2·s −1). These results confirm the validity of the model generally accepted for the exponential growth process and in particular the existence of up to three subgroups of PLL chains from the point of view of their diffusion coefficient. [ABSTRACT FROM AUTHOR]