Response of aquatic organisms to pollutants has been studied following different biomarkers in feral fish at different locations of the Moselle River (France). Both ethoxyresorufin O-deethylase (EROD) and acetylcholinesterase (AChE) activities were measured respectively in the liver and muscle of chub (Leuciscus cephalus) caught by electrofishing. Parallel to this, level of DNA damage (in terms of DNA single strand breaks) was quantified in chub erythrocytes applying the comet assay. Results show a strong difference between males and females in consideration of EROD activity exhibiting 10 fold-values in males compared to females, but not for DNA damage and AChE activity. Moreover response of the 3 biomarkers does not depend on size of fish (highly correlated to their age). EROD activity appears to be induced in males and females according to a growing gradient between upstream and downstream stations but level of DNA damage follows an inverse tendency (i.e. fish from upstream stations exhibit a higher DNA damage than those downstream). Chemical analyses of organic and metallic compounds both in fish muscle and sediment do not clearly explain the responses of studied biomarkers. This study points out the difficulty to assess effects of pollutants in the case of complex contamination and highlights the necessity to use a large array of biomarkers at different levels of biological organisation.