Metals present one of the major contamination problems for freshwater systems, such as the Sava River, due to their high toxicity, persistence, and tendency to accumulate in sediment and living organisms. The comprehensive assessment of the metal bioavailability in the Sava River encompassed the analyses of dissolved and DGT-labile metal species of nine metals (Cd, Co, Cr, Cu, Fe, Mn, Ni, Pb, and Zn) in the river water, as well as evaluation of accumulation of five metals (Cd, Cu, Fe, Mn, and Zn) in three organs (liver, gills and gastrointestinal tissue) of bioindicator organism, fish species European chub (Squalius cephalus L.). This survey was conducted mainly during the year 2006, in two sampling campaigns, in April/May and September, as periods representative for chub spawning and postspawning. Additionally, metal concentrations were determined in the intestinal parasites acanthocephalans, which are known for their high affinity for metal accumulation. Metallothionein concentrations were also determined in three chub organs, as a commonly applied biomarker of metal exposure. Based on the metal concentrations in the river water, the Sava River was defined as weakly contaminated and mainly comparable with unpolluted rivers, which enabled the analyses of physiological variability of metal and metallothionein concentrations in the chub organs, as well as establishment of their constitutive levels.