Indigenous specimens of European chub were caught at five sites in Sava River during two successive non-reproductive periods and used as bioindicator organisms for the assessment of metal accumulation. Liver was selected as a target organ and the metal concentrations (Zn, Cu, Fe, Mn, and Cd) were determined in two fractions of hepatic cytosol, namely in untreated (S50) and heat-treated (HT S50) fraction. In addition, the concentrations of total cytosolic proteins were measured in untreated (S50) fractions, whereas the concentrations of specific metal-binding proteins, i.e. metallothioneins, were determined in the heat-treated fractions. Concentrations of Zn and Fe were significantly higher in the untreated compared to heat-treated cytosol, because these metals are associated to the heat-sensitive metalloenzyme and high molecular mass proteins. On the contrary, Cu, Mn and Cd were predominantly present in the heat-treated cytosol, which contains metallothioneins. In both fractions of hepatic cytosol, nonessential metal Cd showed statistically significant increase from the upstream sites (median 5 ng mL(-1)) toward the downstream site Jasenovac (median 11 ng mL(-1)). The metallothionein concentrations, on the contrary, did not differ between sites, probably due to low dissolved and labile concentrations of metals known as metallothionein inducers (Zn, Cu, and especially Cd) in river water. Therefore, Cd concentrations in hepatic cytosol of European chub can be recommended as an early-warning marker of fish chronic exposure to Cd from combined sources, water and ingested food.