The bioaccumulation potential of environmental pollutants is significantly influenced by the organism’s ability to biotransform chemicals. For aquatic animals, like fish, bioaccumulation evaluations have largely focused on the biotransformation capacity of the liver as the organ responsible for a large proportion of biotransformation activity. However, there is limited knowledge regarding extrahepatic biotransformation and the ability of different organs to support biotransformation processes. In the present study, we provide insight into organ-specific biotransformation through evaluation of in vitro phase I and II biotransformation enzyme kinetics, estimation of intrinsic clearance, and the formation of biotransformation products of selected micro-pollutants (e.g. pharmaceuticals and pesticides). Comparative studies using the liver, intestine, gills, and brain of two salmonid species, rainbow trout (Oncorhynchus mykiss) and brown trout (Salmo trutta), suggest that, while the liver displays the highest activity of specific Cytochrome P450 (phase I) and conjugation (phase II) enzymes, significant activity is also observed in other organs, with certain enzymes, such as CYP2B, displaying similar activity as in the liver. Moreover, based on preliminary chemical analysis, it is likely that the organ-specific clearance and biotransformation product formation of pharmaceuticals and pesticides is proportional to the specific activity of enzymes responsible for their biotransformation. These observations have significant implications for environmental hazard and risk assessments, where the consideration of different biotransformation pathways in different organs would be beneficial for predicting the bioaccumulation potential of chemical pollutants.