With the aim of evaluating cause-effect relationships between metal pollution and biofilm structure and function, a translocation study was performed in a metal-polluted stream (the Osor stream “la riera d’Osor”, NE Spain). Biofilm responses were measured as temporal changes in chl-a fluorescence param-eters, pigment composition and diatom species composition. Biofilms from a non-polluted site were translocated to four downstream sites presenting a gradient of metal pollution, corresponding to scenar-ios of low and moderate metal pollution. Metal effects (Zn bioaccumulation, Fe bioaccumulation and Zn in water) were distinguished from other stressors (phosphate concentration in water). Based on the results obtained, a fast bioaccumulation of Zn (after few hours of exposure) was linked to a decrease in photosyn-thetic efficiency and an enhancement of mechanisms of protection through the xanthophyll cycle. After longer exposure, differences in Zn and Fe bioaccumulation and Zn water concentration between sites were linked to diatom community changes; decrease in diatom cell biovolume, along with a decrease in the IPS index of diatoms. Based on our results, we recommend including Zn in the list of priority pollutant substances of the European Water Framework Directive (2000/60/EC), due to its occurrence and toxic-ity. In addition, our study supports the use of chl-a fluorescence parameters together with the analysis of photosynthetic pigments of biofilms as suitable functional endpoints of early toxicity. Furthermore, diatom community studies are recommended for their clear response after chronic exposure. However, new diatom indices beyond the use of the IPS are required to increase their sensitivity to metal pollution, e.g. to identify the damage caused by moderate to low metal pollution.