This study was designed to investigate the effect of the in vivo and in vitro exposure to the organophosphorothioate pesticide diazinon, its metabolite diazinon oxon and diazinon in a commercial formulation on the AChE activity of the freshwater amphipod Gammarus pulex. Only diazinon oxon at concentrations of ≥ 50x103 nM oxon caused a significant inhibition of acetylcholinesterase (AChE) activity in vitro. However, diazinon oxon had no significant impact on the enzyme activity following in vivo exposure. The latter mode of exposure to either ≥ 10 nM of diazinon or ≥ 5 nM of the diazinon in the commercial formulation caused a significant reduction in AChE activity of treated organisms. This study supports the finding of previous studies that suggested AChE activity in aquatic animals as a valuable biomarker of diazinon.