Science of the Total Environment By measuring levels of tolerance to toxicants in microbial communities using functional toxicity tests under controlled conditions, pollution-induced community tolerance (PICT) approaches offer an effect-based tool to assess the ecological risk of chemicals in aquatic systems. However, induced tolerance of exposed microbial communities cannot always be attributed solely to the presence of toxicants as various environmental factors, such as temperature, can also be involved. Several PICT studies have been conducted to assess the effects of copper (Cu) on phototrophic periphyton, but little is known about the influence of temperature on the response of these microbial communities to acute and chronic exposure to Cu. Here, we report on a microcosm approach to assess the effects of two contrasting temperatures (18°C and 28°C) on (i) the baseline level of Cu tolerance in non-Cu-exposed phototrophic periphyton (i.e. effect of temperature on tolerance baseline), (ii) Cu tolerance acquisition by phototrophic periphyton in response to a 3-week chronic exposure to Cu at a nominal concentration of 60µgL-1 (i.e. effect of temperature on PICT selection) and (iii) tolerance measured during short-term toxicity tests (i.e. effect of temperature on PICT detection). The aim was to evaluate how temperature conditions during the different phases of the PICT approaches may modify the causal relationship between chronic Cu exposure and measured Cu tolerance levels. Our results evidence the influence of temperature both on the basal capacity of phototrophic periphyton to tolerate subsequent exposure to Cu (i.e. influence on tolerance baseline) and on its capacity to acquire tolerance following chronic exposure to Cu (i.e. influence on PICT selection). Hence temperature must be considered when using PICT to establish causal links between chronic Cu exposure and effects on phototrophic periphyton.