Mycobacterium sp VKM Ac-1815D strain was able to cleave sterol side chain giving androstenedione (AD) as a major product with a molar yield of 63-68%. Clones having altered resistance to antibacterial agents were selected. After treatment with ethyl methane sulfonate or mitomycin C, mutants were obtained that retained the ability to produce AD from sitosterol with molar yields of 70-75%. A mutant strain able to effectively reduce 3,17-diketosteroids at C-17 was selected. The 17beta-hydroxy steroid dehydrogenase activity of its crude extract was twice as high as that found for the parent organism. The approach offers the possibility of obtaining improved and labelled biocatalysts for AD or testosterone production from sterols.