In this work, phytosterol-biotransforming strains were selected from Mycobacterium sp., using a high concentration of beta -sitosterol. The selection was made by culturing the strains in a medium enriched with 14 g beta -sitosterol/l as the unique source of carbon. During 2 months, the bacterial cultures were transferred successively. The extraction of the biotrans formation products was made with methanol and ethyl acetate. The qualitative and quantitative analysis was made by means of thin-layer chromatography, gas-liquid chromatography (GLC) and GLC-mass spectrometry. Under these conditions, it was observed that after seven transfers, the strains Mycobacterium sp. MB-3683 and the Mycobacterium fortuitum B-11045 increased their biotransformation capacity from 20% to 64% and from 34% to 55%, respectively. The products in the highest proportion identified for each trial were androstenedione and androstadienedione. The results suggest that the high substrate concentration could be a selective mechanism to obtain strains more efficient in the biotransformation of beta -sitosterol into steroidal bases.