Sertoli cells isolated from 17 day old rats were maintained in culture and incubated with [14C]-progesterone for 20 h. The cells and media were extracted with ether/chloroform and the extracts chromatographed two-dimensionally on TLC and the radioactive metabolites visualized by autoradiography. Nine of the metabolites (constituting about 88% of total metabolite radioactivity) were identified by relative mobilities of the compounds and their derivatives in TLC and GC systems and by recrystallizations with authentic steroids as the following: 20 alpha-hydroxypregn-4-en-3-one, 3 alpha-hydroxy-5 alpha-pregnan-20-one, 5 alpha-pregnane-3 alpha, 20 alpha-diol, 17 beta-hydroxy-5 alpha-androstan-3-one, 5 alpha-pregnane-3,20-dione, 17-hydroxypregn-4-ene-3,20-dione, testosterone, 5 alpha-androstane-3 alpha, 17 beta-diol and androst-4-ene-3,17-dione. Over 71% of the metabolite radioactivity was due to 20 alpha-hydroxypregn-4-en-3-one, the major metabolite. 5 alpha-reduced pregnanes constituted about 12% and C19 steroids comprised about 2.9% of the radioactivity of the metabolites. Calculation of relative steroidogenic enzyme activities from initial reaction rates suggested the following activities in muunits/mg Sertoli cell protein: 20 alpha-hydroxysteroid oxidoreductase (20 alpha-HSO; 7.71), 5 alpha-reductase (4.77), 3 alpha-HASO (3.57), 17 alpha-hydroxylase (0.93), 17 beta-HSO (0.34) and C17-C20 lyase (0.34). The relatively high rate of steroidogenic enzyme activities in the Sertoli cells of young rats may indicate that Sertoli cells are less dependent on Leydig cell steroidogenesis than has been assumed. Since nearly all the metabolites of progesterone and testosterone are now identified, it is possible to construct a picture of Sertoli cell steroidogenic activity.