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Aspergillus niger, one of fungal species most frequently used for experimental and industrial-scale biotransformations of various organic compounds, is generally known to transform steroids at 16β position. In this work, application of the strain A. niger KCH910 to bioconversion of dehydroepiandrosterone (DHEA), androstenediol and testosterone is described, with emphasis on the metabolic steps leading to the products. Evidence from this study indicated that incubated 5-ene steroids underwent bioconversion within two metabolic pathways: oxidation by the action of 3β-HSD (3β-hydroxysteroid dehydrogenase) to 4-ene steroids, and minor allylic hydroxylation to epimeric 7-alcohols. Further transformation of the 3-oxo-4-ene metabolites resulted in non-selective 16-hydroxylation. It is the first report on an A. niger strain able to introduce not only 16β- but also 16α-hydroxyl function into steroids.Testosterone, DHEA, and androstenediol were transformed by A. niger KCH910.3-Oxo-4-ene steroids were hydroxylated at 16α- and 16β-position.This is the first report on 16α-hydroxylation of steroids by A. niger strain.Hydroxylation of 5-ene androstanes led to both 7β- and 7α-alcohols.3β-hydroxy-5-ene steroids were converted by 3β-HSD to 3-oxo-4-ene steroids.