We studied the metallothionein (NIT) response in cadmium-exposed worms (Eisenia fetida) both at the protein level by Dot Immunobinding Assay (DIA) with a polyclonal antibody raised against the most immunogenic part of this protein and at the expression level by Northern blotting using a specific probe. NIT appeared as two close isoforms. DIA results clearly demonstrated significant differences in MT level of whole worm heat-treated supernatants between E. fetida exposed to Cd concentrations as low as 8 mg Cd kg(-1) of dry soil compared to controls. Northern blotting analysis performed on whole bodies of worms revealed that a single exposure to 8 mg Cd kg-1 of dry soil for I day resulted in the production of NIT mRNA. This response was maintained for exposure of at least I month. Clear differences of NIT gene expression were also observed between worms exposed to different Cd concentrations (8, 80 or 800 mg; Cd kg(-1) of dry soil). Immunocytochemistry demonstrated that NIT was located in the chloragogenous tissue surrounding the gut where metals are known to be accumulated. This work revealed that E. fetida NIT is a sensitive and relevant biomarker of Cd exposure and especially when considering gene expression response. Further experiments have now to prove its usefulness in natural metal-contaminated soil toxicity assessments. (c) 2006 Elsevier Inc. All rights reserved.