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Metallothioneins (MT) are a validated biomarker for metal exposure. In fish they are usually determined in the liver, while some studies have observed MT in kidney, muscle and gills. There are, however, no reports on MT in fish bile. This study aimed to verify if these proteins are present in fish bile, if they can be used as biomarkers for metal exposure and to characterize the metal speciation present in the different species. Three independent methods to verify MT presence in fish bile were conducted: 1D/2D gel electrophoresis, spectrophotometric analyses and SEC-HPLC–ICP-MS. Results show that all methods verified the presence of these proteins in fish bile, while SEC-HPLC–ICP-MS also characterized also characterized metals responsible for the contamination of the studied sites. Quantification was successfully conducted by two methods, densitometry (1D electrophoresis) and spectrophotometry, and the …