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Fast-growing mycobacteria such as Mycobacterium sp. and Mycobacterium smegmatis degrade natural sterols. They are a model to study tuberculosis. Interestingly, M. smegmatis has been found in river effluents derived from paper production, and therefore, it would be important to gain further insight into its capacity to synthesize steroids that are potential endocrine disruptors affecting the development and reproduction of fishes. To our knowledge, the capacity of M. smegmatis to synthesize estrogens and even testosterone has not been previously reported. Therefore, the objective of this study was to investigate the capacity of M. smegmatis to synthesize in vitro testosterone and estrogens from tritiated precursors and to investigate the metabolic pathways involved. Results obtained by thin-layer chromatography showed that3H-progesterone was transformed to 17OH-progesterone, androstenedione, testosterone, estrone, and estradiol after 6, 12, or 24 h of incubation.3H-androstenedione was transformed into testosterone and estrogens, mainly estrone, and3H-testosterone was transformed to estrone and androstenedione. Incubation with3H-dehydroepiandrosterone rendered androstenediol, testosterone, and estrogens. This ability to transform less potent sex steroids like androstenedione and estrone into other more active steroids like testosterone and estradiol or vice versa suggests that M. smegmatis can influence the amount of self-synthesized strong androgens and estrogens and can transform those found in the environment.