The composition of steroid metabolites formed during the conversion of androstenedione and androstadienedione, products of degradation of sterol side chains by soil and mutant strains of the bacterial genera Mycobacterium and Protaminobacter, was studied. Testololactone was absent from the conversion products. This favors the idea of different cleavage pathways of steroid ring D in bacteria and fungi. Very small amounts of two new 14alpha-hydroxy derivatives with cleaved B ring were isolated after conversion of androstenedione by soil strains. It was shown that a mutant Mycobacterium smegmatis strain, as well as wild strains, could perform 14alpha-hydroxylation of steroids. It is suggested that cleavage of the steroid nucleus at the side of rings D and C starts with the introduction of a 14alpha-hydroxy group followed by dehydration.