Steroids undergoing enterohepatic circulation are exposed to bacterial metabolism particularly by obligate anaerobes which account for 99.99% of the fecal flora. The most common transformation is hydrolysis of conjugated steroids. The glucuronidases are synthesized by Escherichia coli and Bacteroides species. The bacterial catabolism of unconjugated steroids may be considered under several headings: 1. Reduction of ring-A due to clostridia species synthesizing specific enzymes; C. paraputrificum, 3 alpha,5 beta-reductase; C. innocuum, 3 beta,5 beta-reductase; and a new species C.J-1, 3 beta,5 alpha-reductase. 2. Reduction of the delta 5 bond by human fecal flora. The specific strain(s) synthesizing the enzyme have not yet been identified. 3. Reduction of 17-keto estrogens by the above mentioned ring-A reducing clostridia and by Eubacterium lentum. 4. Reduction of 17-keto androstenes by Bacteroides fragilis. 5. Desmolase mediated side chain cleavage at C17-C20 position of 17 alpha-hydroxysteroids by two new species Clostridium scindens and Eubacterium desmolans isolated from human and cat fecal flora respectively and by Clostridium cadavaris isolated from New York City sewage. 6. And 16 alpha- and 21-dehydroxylase by E. lentum a normal inhabitant of the human gut; it is the only organism known to synthesize 16 alpha- or 21-dehydroxylases. Due to the high specificity of the enzymes and the simplicity of extracting the metabolites, biosynthesis of reference compounds and radioimmunoassay reagents is practical and inexpensive. The enzymes can also be used for titration of specific bacterial strains in fecal flora and as markers for bacterial identification in particular for the strains difficult to be defined by regular biochemical reactions.