Variations in (a) carbon and nitrogen stable isotope composition (as δ13C and δ15N), and (b) trace metal content of sediments and biota (algae, invertebrates and fish) were investigated at four sites along the river Po (from the middle reaches to the delta) and one site in the coastal Adriatic area influenced by the river. The aim was to begin accumulating a database to help interpret differences in the accumulation of toxic substances in biota and sediments, elucidating how food sources and feeding strategies change between areas of the system differing in terms of anthropogenic influence and distance from the river mouth. C content and δ13C and δ15N were determined on biological tissue and sediment samples using a continuous flow isotope ratio mass spectrometer. Trace metal concentrations were assayed by electrothermal (Cd, Cu, Cr, Ni, Pb) or flame (Zn) atomic absorption spectrometry after acid digestion. Hg was assayed on solid samples on an automated mercury analyser using the vapour generation technique. In the sediments, depletions of the 13C isotope at the post-Lambro site (downstream) relative to the upstream Lambro site were indicative of the significant pollutant input from this tributary to the Po. Mercury, cadmium and zinc concentrations in sediments were two-to-three times higher downstream compared to upstream. Differences in carbon and nitrogen isotope signatures in fish species among the two Lambro sites and the last stretch of the Po indicated variations in the type of food and primary carbon sources. Only for chromium were concentrations significantly higher in fish muscle from the post Lambro site than from upstream of the Lambro or the last reaches of the Po. Mean levels of cadmium and zinc in the muscle of omnivorous fish at the four river sites were low and similar (< 0.01 to 0.02 μg g-1 for Cd, 22–31 μg g-1 for zinc), while between-site differences were found for the other metals. Only for mercury did bioaccumulation reflect trophic level movement.