Aromatase, a key steroidogenic enzyme that catalyses the conversion of androgens to estrogens, represent a target for endocrine disrupting chemicals. However, little is known about the effect of pollutants on aromatase enzymes in fish. In this study, we first optimized a rainbow trout (Oncorhynchus mykiss) microsomal aromatase assay to measure the effects of 43 substances belonging to diverse chemical classes (steroidal and non steroidal aromatase inhibitors, pesticides, heavy metals, organotin compounds, dioxins, polycyclic aromatic hydrocarbons) on brain and ovarian aromatase activities in vitro. Our results showed that 12 compounds were able to inhibit brain and ovarian aromatase activities in a dose-dependent manner with IC50 values ranging from the low nM to the high microM range depending on the substance: steroidal and non steroidal inhibitors of aromatase (4-hydroxyandrostenedione, androstatrienedione, aminogluthethimide), imidazole fungicides (clotrimazole, imazalil, prochloraz), triazole fungicides (difenoconazole, fenbuconazole, propiconazole, triadimenol), the pyrimidine fungicide fenarimol and methylmercury. Overall, this study demonstrates that rainbow trout brain and ovarian microsomal aromatase assay is suitable for evaluating potential aromatase inhibitors in vitro notably with respect to environmental screening. The results highlight that methylmercury and some pesticides that are currently used throughout the world, have the potential to interfere with the biosynthesis of endogenous estrogens in fish.