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The aim of the present study was to assess aquatic contamination by polycyclic aromatic hydrocarbons (PAH), using the 1-hydroxypyrene (1-OHP) content in fish bile as a biochemical marker. A total of 71 chub (Leuciscus cephalus L.) were collected from seven locations on the Svitava and Svratka rivers in and around the industrial city of Brno, Czech Republic. The levels of 1-OHP were determined by reverse phase HPLC with fluorescence detection after deconjugation. Normalising the molar concentration of the biliary 1-OHP to the biliary protein content reduced sample variation. The content of 1-OHP was correlated with the PAH level in bottom sediment and semi-permeable membrane devices (SPMD), which was analyzed by a combination of HPLC/FLD and GC/MS methods. The highest mean values of 1-OHP were found in fish caught at the Svratka River at locations Modrice (169.2 ± 99.7 ng•mg-1 protein) and Rajhradice (152.2 ± 79.7 ng•mg-1 protein), which are located downstream from Brno. These values were significantly (P < 0.05) higher than those obtained from localities Knínicky (98.4 ± 66.1 ng•mg-1 protein) and Bílovice nad Svitavou (64.1 ± 31.4 ng•mg-1 protein). The lowest contents of PAH in sediment and SPMD were found at location Knínicky (1.5 mg•kg-1 dry mass and 19.4 ng•L-1, respectively). The highest contents of PAH in sediment and SPMD were found in Rajhradice (26.0 mg•kg-1 dry mass) and Svitava before junction (65.4 ng•L-1), respectively. A Spearman correlation test was applied to determine the relationship between biliary 1-OHP and the sum of PAH in sediment and SPMD. A positive, but no statistically significant correlation was found. The main impact sources of elevated level of PAHs in sites located downstream from Brno are most probably intensive industrial and agricultural activities and domestic waste. Full article