Abstract Two human intestinal anaerobes, culture No. 116 and Eubacterium lentum, synthesize enzymes capable of metabolizing C-21 steroid hormones. 21-Dehydroxylase activity by culture No. 116 benefits from the presence of Escherichia coli or allied organisms in the medium; their effect appears to be a lowering of the Eh. The enzymes activity is independent of arginine and shows no sign of product inhibition. The enzyme, which is constitutive, functions at pH 6.5–7.3; its activity is unrelated to the 4-ene-3CO group but is inhibited by a C20-OH group. E. lentum (neotype), in addition to a 21-dehydroxylase, synthesizes a 3α-hydroxysteroid dehydrogenase active at Eh ± —150 mV, which explains the epimerization of 3α-pregnanolone observed in cultures of fecal flora. The metabolic alterations of deoxycorticosterone by cultures of fecal flora may be duplicated by Clostridium paraputrificum and E. lentum, whether acting in concert or sequentially.