Carboxylesterases (CbEs) are key enzymes in pesticide detoxification. These esterases are involved in the biochemical mechanismfor pesticide resistance in some pest species, and further they are considered an efficient protective mechanism against acute toxicity by organophosphate (OP) pesticides in mammals. To gain knowledge on the role of CbEs in pesticide toxicity and natural tolerance in earthworms, we performed an enzyme kinetic analysis to investigate whether these annelids are able to secrete them into their gut lumen. We determined levels of CbE activity and isozyme abundance in the gutwall and ingested soil collected fromdifferent portions of the gastrointestinal tract of Lumbricus terrestris. Moreover, modulation of enzyme activity by selected substrates (α-naphthyl acetate [α-NA], 4-nitrophenyl valerate [4-NPV] and 4-nitrophenyl acetate [4-NPA]) and OP pesticideswas examined to compare the response between tissue and soil CbEs.Wefound a high CbE activity in the ingested soil extracts from the crop/gizzard (α-NA-CbE=8.43±2.76 U mg−1 protein and 4-NPACbE= 5.98±2.11 U mg−1 protein) compared to the gut wall. Three lines of evidences suggest that the gut epithelium is the main source of this luminal CbE activity. First, the effect of substrate concentrations on CbE activity from both the ingested soil extracts and gut tissues resulted in similar apparent Km and Vmax values. Second, native PAGE gels revealed that some of the CbE isozymes in the gut tissue were also present in the soil extracts. Third, tissue and soil CbEs showed the same sensitivity to inhibition by OPs. The concentrations of insecticide causing 50% of esterase inhibition (IC50)was comparable between tissue (IC50s range=4.01–9.67 nM dichlorvos and 8480–6880 nM paraoxon) and soil (IC50s range=6.01–11.5 nM dichlorvos and 8400–7260 nM paraoxon). Our results suggest a set of (eco)toxicological implications and environmental applications derived from the ability of earthworms to secrete these pesticide-detoxifying enzymes