Gudgeons (Gobio gobio) from historically Cd and Zn contaminated sites in Flanders (Belgium) were found to be resistant to elevated Cd levels. In previous work, this increased resistance was largely explained by increased metallothionein (MT) expression. Recently, environmental cleanup efforts resulted in a significant decrease in Cd concentrations in the surface water. In this study, we evaluated the use of hepatic metal and metallothionein (MT) concentrations as biomarkers of metal exposure before and after the cleanup. Hepatic MT mRNA levels were determined after the environmental metal levels decreased in order to assess the applicability of MT gene expression as an environmental biomarker in natural fish populations. Our data show that both metallothionein protein and gene expression have the potential to be sensitive biomarkers for metal exposure. Significant correlations were found (a) among accumulated metal concentrations and both MT protein and mRNA levels, and (b) between MT protein and mRNA levels. However, our data illustrated that while MT protein and gene expression give a quantitative picture of metal load at a single time point, quantitative information in natural populations cannot always be obtained when different time points (including different years) are compared, since MT gene and protein expression are affected by many other factors in addition to the metal load. Furthermore, the result of the environmental cleanup was reflected in a decrease of hepatic Cd concentrations. Zn remained the most important factor determining MT concentrations. Finally, two differently sized MT mRNAs were amplified to test the hypothesis that 3'-UTR length can offer a protective advantage in conditions of environmental stress. Our data provided no evidence to support this hypothesis. In contrast, the ratio of the long mRNA variant relative to total MT mRNA was surprisingly constant, and independent of exposure history.