Mucor piriformis was used to study the mode of transformation of 16-dehydroprogesterone (I, pregna-4, 16-diene-3, 20-dione) and 17α-hydroxyprogesterone (II, 17α-hydroxypregn-4-ene-3, 20-dione). Biotransformation products formed from I were 14α-hydroxypregna-4, 16-diene-3, 20-dione (Ia), 7α, 14α-dihydroxypregna-4, 16-diene-3, 20-dione (Ib), 3β, 7α, 14α-trihydroxy-5α-pregn-16-en-20-one (Ic), and 3α, 7α, 14α-trihydroxy-5α-pregn-16-en-20-one (Id). Metabolites Ic and Id appear to be hitherto unknown. Time-course studies suggested that the transformation is initiated by hydroxylation at the 14α-position (Ia) followed by hydroxylation at the 7α-position (Ib). Microsomes (105,000 g sediment) prepared from 16-dehydroprogesterone-induced cells hydroxylate I to its 14α-hydroxy derivative (Ia) in the presence of NADPH. Incubation of Ia with the organism resulted in the formation of Ib, Ic and Id. Biotransformation products formed from compound II were 17α, 20α-dihydroxypregn-4-en-3-one (IIa), 7α, 17α-dihydroxypregn-4-ene-3, 20-dione (IIb), 6β, 17α, 20α-trihydroxypregn-4-en-3-one (IIc) and 11α, 17α, 20α-trihydroxypregn-4-en-3-one (IId). Time-course studies indicated that IIa is the initial product formed, which is further hydroxylated either at the 6β or 11α position. Incubation of IIa with the organism resulted in the formation of IIc and IId. Reduction of the 4-en-3-one system and 20-keto group has not been observed before in organisms of the order Mucorales. In addition, M. piriformis has been shown to carry out hydroxylation at the C-6, C-7, C-11 and C-14 positions in the steroid molecules tested.