Using newborn rat adrenal cells in primary culture, 16 alpha-hydroxyprogesterone was bioconverted into numerous 16 alpha-hydroxylated steroids. The method of analysis of these steroids comprised the association of column and thin-layer chromatography to gas chromatography-mass spectrometry in order to obtain the mass spectra of pure compounds. The identified compounds resulted principally from the enzymatic reactions of 21-hydroxylation 11 beta-hydroxylation and reduction of the 20-oxo and 3-oxo-4-ene groups. Minor metabolites resulted from 18-hydroxylation and 6 beta-hydroxylation of the substrate. The metabolism of 16 alpha-hydroxyprogesterone is similar to that of progesterone in the same cell-culture system; however, there are two exceptions. The 21-hydroxylation of 16 alpha-hydroxyprogesterone occurs at a rate similar to that of its 11 beta-hydroxylation, whereas the 21-hydroxylation of progesterone is faster than its 11 beta-hydroxylation. The ratio of 11 beta- to 18-hydroxylation of 16 alpha-hydroxyprogesterone is about 3, whereas the ratio of 11 beta- to 18-hydroxylation of progesterone, 20 alpha-dihydroxyprogesterone and DOC is between 1./ and 2. It is most likely the rate of 18-hydroxylation which is decreased by the hydroxyl group at C-16. The use of adrenal cell cultures is a practical, simple method for the preparation of a variety of 16 alpha-hydroxylated steroids from a single substrate. Its adaptation to the production of important amounts of 16 alpha-hydroxylated corticosteroids will permit the study of their biological activity.