River biofilms are exposed to various contaminants leading to their bioaccumulation within this biological matrix. The different species of the biofilms are included in an extracellular polymeric substance (EPS) matrix, potentially protecting them from the penetration of contaminants. Several studies have suggested that during biofilm growth, biofilm and EPS matrix thickness would "protect" bioaccumulation cells (e.g. Ivorrra et al., 2000; Duong et al., 2010). To demonstrate this hypothesis, we conducted an experiment under controlled conditions consisting to grow biofilm in the early stages of its development (0 to 20 days) in water spiked with natural dissolved copper. Then, we transferred this biofilm to a mono-isotopic (65Cu) copper-enriched medium for 20 additional days. During these two exposure periods, dissolved copper concentrations and the corresponding 63Cu/65Cu isotopic ratios were monitored. At the end of the 2 different exposure periods, the glass slides were scraped to recover and freeze-dried the biofilm. A sequential extraction was then applied on the biofilm to recover copper from the colloidal and capsular EPS fractions. The pellet was then mineralized to determine copper concentrations in the intracellular fraction. Copper concentrations and isotopic ratios were determined in the water and in the different fractions of the biofilm by ICP-MS. The results showed that we were able to maintain constant copper concentrations during the two exposure periods (~7 µg/L) while isotopic ratios 63Cu/65Cu were well contrasted between the first (2.23) and the second phase (0.25) of exposure. Results of Cu concentrations in the different biofilm fractions showed that bioaccumulation in the young biofilm were similar between the colloidal, capsular and cellular fractions. Finally, the isotopic approach showed that after 40 days of exposure, the isotopic ratios in the different fractions of the biofilm were similar to the ratio in the dissolved media of the second phase of exposure (~0.25). These results suggest (i) an intense and rapid renewal of the biofilm and bioaccumulated Cu and (ii) that Cu concentrations in a mature biofilm at a given time reflects the last days of exposure. In addition, although the ratios were very low, a significant difference of isotopic ratios between the EPS fraction (0.25) and the cell fraction (0.35) confirms the potential protective function of the EPS matrix.