To validate cholinesterase activity as a biomarker of pesticide exposure, we characterized the tissue distribution (whole body, nervous tissue and crop/gizzard), activity at two seasons of cholinesterase in six different species of earthworms collected in an unpolluted field: Lumbricus terrestris, Lumbricus castaneus, Aporrectodea nocturna, Aporrectodea caliginosa, Allolobophora chlorotica and Aporrectodea rosea. The major part of total cholinesterase activity was found in the nervous tissue while activity in crop/gizzard was weak. The level of the total cholinesterase activity was stable for each species considered throughout the year (spring and autumn). Lumbricus species exhibited three-fold higher specific activity than the others (0.086±0.015 U mg−1 and 0.235±0.036 U mg−1 for Allolobophora or Aporrectodea, and Lumbricus species respectively). This stability of the base level makes cholinesterase activity a useful biomarker for monitoring effects of pesticide under natural conditions. Cholinesterase activity was characterized using different substrates and inhibitors. It seems likely that the cholinesterases are acetylcholinesterases in most species investigated as they preferentially hydrolyzed acetylthiocholine and were inhibited by eserine, but not by tetraisopropyl pyrophoramide (iso-OMPA). Characterization of cholinesterase from Allolobophora chlorotica is uncertain and it cannot be classified as a true AChE.