Metallothionein is a key, well characterized metal binding protein in mammalian systems. Its chemical properties, proposed functions, and regulation of transcription by diverse agents are critically reviewed with the objective to relate this information to the proposed use of metallothionein as a biomarker. Careful examination is given to several alternative methods for quantitation of this protein. Then, specific results from the authors’ laboratories are used to consider in more detail questions involved in understanding the meaning of measurement of metallothionein or other metal binding proteins or peptides in non-mammalian, aquatic systems including fish, anurans, and microorganisms. It is shown that before such determinations gain meaning, one must know whether measured levels of binding protein represent basal concentrations or induction of protein after exposure to metals, host response to stress, or a complex combination of them. Finally, it is concluded that if metallothionein-like structures are to serve as biomarkers in aquatic systems, much more direct study of specific aquatic organisms will be needed.