Equilibrium sampling devices (ESDs) can be applied to study and monitor the exposure and fate of hydrophobic organic chemicals (HOCs) on a thermodynamic basis. ESDs can be used to determine freely dissolved concentrations and chemical activity ratios, and to predict equilibrium partitioning concentrations of HOCs in biota lipids. Our aim was to assess the equilibrium status of polychlorinated biphenyls (PCBs) in a contaminated lake ecosystem and along its discharge course by using ESDs for measurements in sediment and water, and by additionally analysing biota. We used ESDs (silicone rubber and polyethylene) to determine freely dissolved concentrations and chemical activities of PCBs in the water column and sediment pore water, and calculated for both phases the corresponding equilibrium concentrations and chemical activities in model lipids. Overall, the studied ecosystem appeared to be in disequilibrium for the studied phases; sediment, water and biota. Chemical activities of PCBs were higher in sediment than in water, which implies that the sediment functioned as a partitioning source of PCBs and that net diffusion occurred from the sediment to the water column. Measured lipid-normalized PCB concentrations in biota were generally below equilibrium lipid concentrations relative to the sediment (CLip?Sed) or water (CLip?W), indicating that PCB levels in the organisms were below the maximum partitioning levels. This study showed the application versatility of ESDs in the field, and facilitated a thermodynamic understanding of exposure and fate of PCBs in a contaminated lake and its discharge course.