When [4-14C]-5 alpha-dihydrotestosterone was incubated with the homogenate of human epididymis, 5 alpha-androstane-3 alpha, 17 beta-diol and 5 alpha-androstane-3 beta, 17 beta-diol were identified as major metabolites. The ratio of 3 alpha- to 3 beta-epimer in androstanediol formation was approximately 2.4. 5 alpha-Androstane-3, 17-dione was also identified as a minor metabolite. Among the subcellular fractions, both the human epididymal 3 alpha- and 3 beta-hydroxysteroid dehydrogenases were localized almost exclusively in the cytosol fraction (105,000 X g supernatant). Both enzymes had optimum pH at 7.5 and optimum temperature at 46 degrees C. NADPH was a more preferable cofactor than NADH for both dehydrogenases. The Michaelis constants (Km) of 3 alpha- and 3 beta-hydroxysteroid dehydrogenase for 5 alpha-dihydrotestosterone were similar and estimated as 8 X 10(-5) M, but the enzymes were unsaturable with the substrate under the conditions investigated, indicating low affinity and high capacity of both dehydrogenases for 5 alpha-dihydrotestosterone. The human epididymal 5 alpha-reductase revealed a regional difference in activity. The 5 alpha-reductase activity in the most proximal part of the head (ductuli efferentes) was one seventh to one tenth the activity in the remaining part of the epididymis which was constructed of ductus epididymis. Except for this finding, the activity of 5 alpha-reductase was highest in the head, then declined along the course to the tail portion. The 5 alpha-reductase for testosterone was competitively inhibited by delta 4-3-oxosteroids such as progesterone, 20 alpha-dihydroprogesterone, 17 alpha-hydroxyprogesterone, 4-androstenedione, 11-deoxycorticosterone, corticosterone and 11-deoxycortisol, which had inhibition constants (Ki) of 3.3 X 10(-9) M, 2.2 X 10(-9) M, 1.8 X 10(-8) M, 1.3 X 10(-8) M, 8.3 X 10(-9) M, 1.5 X 10(-7) M and 8.7 X 10(-8) M, respectively, suggesting the possibility that the 5 alpha-reduction of testosterone is regulated by other delta 4-3-oxosteroids.