Abstract The biomarker concept is an important approach of modern ecotoxicology based on the detection and quantification of molecular, biochemical, cellular, or physiological alterations in organisms as the result of exposure to toxic compounds. In the present study, the biomarker potential of two metallothionein (MT) isoforms from hepatopancreas and mantle tissue of the Roman snail, Helix pomatia L., is evaluated. This species possesses two MT isoforms with specific functions: The Cd-specific isoform (Cd-MT) is predominantly induced and expressed in the hepatopancreas upon Cd exposure. It binds and inactivates this potentially toxic trace element. The second isoform is Cu-specific (Cu-MT), primarily expressed in the mantle tissue and responsible for the homeostatic regulation of Cu. The differential expression and metal-specificity of the two isoforms make them potential candidates for a dual MT biomarker system based on an isoform-specific approach. This study shows that Cd-MT and Cu-MT of H. pomatia can reliably be quantified by a differential metal saturation approach that could be used for environmental biomarker studies. The approach works by virtue of the following preconditions: The two isoforms differ from each other with respect to their molecular and biochemical features, they show metal-specific binding preferences, they are organ-specific, they exhibit distinct induction patterns in response to the metals to which they bind, and their biological and functional background is thoroughly known.