We report here the structural assignment for the hydroxylated 5 alpha-reduced metabolites in the culture medium following incubation of radiolabelled testosterone with explants of rat ventral prostate, canine cauda epididymidis and prostate, and benign hyperplastic canine and human prostate. Explants were incubated for 21 h at 37 degrees C in surface contact with serum-free Trowell's T8 medium containing 1.7 microM or 8.5 nM substrate. After uptake determination, radiosteroid patterns in explants and media were obtained by t.l.c. The C19O3-metabolites released into the culture medium were resolved by h.p.l.c. and fractions migrating with appropriate reference compounds were crystallized to constant SA with carriers synthesized for this purpose. 5 alpha-Androstane-3 beta,6 alpha,17 beta-triol and 3 beta,6 alpha-dihydroxy-5 alpha-androstan-17-one were identified as the major C19O3-radiometabolites of rat ventral prostate. In the culture medium of canine prostate and epididymis and human prostate, 5 alpha-androstane-3 beta,7 alpha,17 beta-triol and 3 beta,7 alpha-dihydroxy 5 alpha-androstan-17-one were the principal hydroxylation products, with 6 alpha-hydroxy epimers as significant minor products of the canine prostate and epididymis and 5 alpha-androstane-3 beta,7 beta,17 beta-triol as a significant minor radiometabolite of human prostate tissue. Treatment of the castrated dog with androgen and estrogen causes an oxidative shift to striking predominance of 3 beta,7 alpha-dihydroxy 5 alpha-androstan-17-one. The 3 beta-hydroxy-5 alpha-androstane configuration of the identified C19O3-metabolites supports a pathway of prostatic and epididymal testosterone disposition which effects activation by 5 alpha-reductase and inactivation and egress by the coupled 5 alpha-3-oxosteroid reductase/3 beta-hydroxysteroid hydroxylase reactions.