Biomarkers in fish may serve as a useful tool for evaluating the pollution load in the environment and for early warning signals about new environmental threats. By employing a strategy with fish that are caged or reared in tanks, problems linked to migration and feeding status can be reduced or eliminated. Such a strategy, however, also may introduce other confounding factors linked to, for example, hierarchical behavior or disease outbreaks. In the present study, rainbow trout (Oncorhynchus mykiss) were reared in plastic tanks at four sites in the Göta älv river system (plus one external reference) in western Sweden during 2006 and 2007. Because of low population density and high water turnover in the area, pollution levels are expected to be low. Therefore, this should be a good test for the sensitivity of the methodology. Several significant differences were found between sites as well as between years, such as ethoxyresorufin-O-deethylase (EROD) activity, polycyclic aromatic hydrocarbon (PAH) metabolites in bile, and concentration of inorganic ions in blood plasma, but it also was seen that factors other than pollution could be contributing to these differences. The condition factor (CF) varied between sites, possibly because of differences in feeding resulting from variations in water turbidity. Furthermore, even the small differences in CF that were found within sites correlated significantly to several of the biomarkers. It was shown that PAHs likely are the most important EROD inducers in Göta älv and that variation over time is greater than variation between sites. Because CF differed between sites despite a standardized feeding ration, starving of the fish during the exposure period should be considered for future studies.