The response of wild chubs (Leuciscus cephalus) to chemical pollution was assessed in a metal contaminated river (Cecina River, Italy) through a wide battery of biomarkers which included: Comet assay detecting DNA strand breaks; diffusion assay for apoptosis induction; micronucleus test assessing chromosomal alterations; ethoxyresorufin O-deethylase (EROD) activity for the induction of cytochrome P 4501A; acetylcholinesterase (AChE) activity responsive to pesticide exposure; vitellogenin gene expression in males revealing estrogenic effects. Bioaccumulation of mercury, chromium and polycyclic aromatic hydrocarbons (PAHs) was also determined. Levels of mercury and PAHs were higher in tissues of chubs sampled from the most downstream station, reflecting an anthropogenic pollution of industrial origin. Otherwise, accumulation of Cr was quite similar in fish along the entire course of Cecina River confirming a natural origin due to local geochemical features. Biomarker responses revealed a significant increase of apoptotic cells, DNA stand breaks and micronucleus frequency in chubs from the more impacted sites. A slight EROD induction and AChE inhibition were only seen at the most downstream station demonstrating a limited impact due to PAHs and pesticides. On the other hand, the induction of vitellogenin gene in male chubs was measured in all the sites, suggesting a diffuse estrogenic effect. This study confirmed the utility of large batteries of biomarkers in biomonitoring studies and the suitability of wild chub as bioindicator organism for river basins.