The aim of this study was to characterize cholinesterase (ChE) activity in Gammarus pulex, an abundant and ecologically relevant species of the European stream environment. Biochemical and pharmacological properties were tested using different substrates (acetylthiocholine iodide, propionylthiocholine iodide and butyrylthiocholine iodide) and selective inhibitors (eserine sulfate, BW284c51 and iso-OMPA). In a second part, the in vitro and in vivo effects of a widely used organophosphorous pesticide, chlorpyrifos, on ChE activity were investigated. The results suggest that G. pulex possess only one ChE which displays the typical properties of an acetylcholinesterase, since: (1) it hydrolyses to the substrate acetylthiocholine at a higher rate than all other tested substrates and (2) it is highly sensitive to eserine sulphate and BW284c51, but not to iso-OMPA. In vitro and in vivo inhibitions were observed for highly different contamination levels, which suggests that bioaccumulation and biotransformation mechanisms are involved. In vivo AChE inhibition was observed at realistic environmental concentrations, with lethal effects appearing at inhibitions higher than 50%. The results of this study show the value of G. pulex as a sentinel organism for environmental assessment.