The side chain of certain C-21 steroids may be removed by an enzyme, desmolase, synthesized by intestinal bacteria. With a view to isolate these organisms we examined the conditions required for their multiplication and function. The model substrate, cortisol (11 beta, 17 alpha, 21-trihydroxy-4-pregnene-3,20-dione), was metabolized by mixed fecal flora of humans and rats to a number of C-21 and C-19 compounds. The major C-21 metabolites were 21-deoxycortisol, tetrahydro-21-deoxycortisol, and tetrahydrocortisol. The C-19 metabolites obtained were identified as 5 beta-androstane-3 alpha, 11 beta-triol and 5 xi-androstane-3 alpha, 11 beta-diol-17-one. The prevalence of converting microorganisms was approximately 10(6)/g feces in both humans and rats. Conversion required an Eh below -130 mV, and an initial pH of 7.0. Optimal yield of C-19 products occurred with a fecal dilution of 10(5), though C-19 metabolites were evident from 10(1) through 10(8) fecal dilutions. Preliminary investigation indicates that the ability of converting organisms to form colonies varied with the composition of the media and the gaseous environment.