As a widely spread cyprinid fish species, the European chub (Leuciscus cephalus) has been used extensively in bio-monitoring programs. However, no laboratory dose-response and/or time course studies related to applied biomarkers have been reported on chub yet. In order to address this issue, specimens of juvenile chub caught in September 2005 in the Sava River, Croatia, were laboratory exposed to various (0.25–50 mg/kg) doses of either model polycyclic aromatic hydrocarbons (PAH) premutagen benzo[a]pyrene (BaP), or β-naphthoflavone (β-NF), a well-known model cytochrome 1A (CYP1A) inducer, for 3 (BaP) or 5 d (β-NF). The responses of several hepatic biomarkers were determined in the exposed fish: The hepatic 7-ethoxyresorufin-O-deethylase activity, CYP1A content, glutathione S-transferase (GST) activity, liver bioactivation potential, and the amount of hydroxylated polycyclic aromatic hydrocarbon bile metabolites determined by the fixed wavelength fluorescence and the high-performance liquid chromatography technique. The relevance of determined biomarker responses has been analyzed further and crosscorrelated with the same set of biomarkers, as well as with tissue concentrations of polycyclic aromatic hydrocarbons and polychlorinated biphenyls, determined in chub specimens collected in September 2005 at five different polluted locations along the Sava River. The species-specific upper and lower limits in responses of studied biomarkers were determined and the obtained ranges successfully evaluated in real field situation. With the exception of the GST activity, all other biomarkers determined in chub proved to be valuable indicators of environmental pollution. Finally, the results of the present study demonstrated that the same strategy of laboratory characterization in combination with field evaluation should be used regularly in the selection of optimal biomarkers and indicator species.