Androgen biosynthesis in the testis may be analyzed in some detail by means of techniques of in vitro incubation of small testicular biopsy specimens with suitable radiolabelled precursors. Sixty-six tissue specimens from 33 patients who underwent bilateral testicular biopsies because of infertility were incubated in vitro with [3H]pregnenolone in order to investigate the possibility of abnormalities in their steroid biosynthetic activity. As a normal control, testicular tissue obtained by testicular biopsy from a young normal volunteer was used. The distribution of metabolites in the incubates of testes from 8 infertile men differed greatly from the remaining 25 patients and the normal control. The major steroids formed from pregnenolone by the testes of those 8 men were 17-hydroxypregnenolone, dehydroepiandrosterone, 20α-dihydropregnenolone and 20α-dihydro-17-hydroxypregnenolone. Very small amounts of Δ4-3 oxo products (progesterone, 17-hydroxyprogesterone, androstenedione and testosterone) were formed suggesting a deficiency of 3β-hydroxy-steroid-dehydrogenase activity in the testes of these 8 men, possibly related to the derangement of their spermatogenic function.