Thirty-four incubations of minced testis tissue from five patients with prostatic carcinoma were performed for different periods of time with labeled precursors pregnenolone, progesterone, 17-hydroxypregnenolone, 17-hydroxyprogesterone, dehydroepiandrosterone (DHA), androst-4-ene-3,17-dione (Δ4-dione), and androst-5-ene-3β, 17β-diol (Δ5-diol) singly or in varying combinations. Testosterone (T) and the Δ5 and Δ4 intermediates which have been suggested as possible precursors of T were identified. Metabolism of pregnenolone or 17-hydroxypregnenolone yielded steadily increasing T with a bell shaped curve depicting the Δ5 intermediates, whereas the recovery of Δ4 intermediates remained low throughout the incubation period. From a comparison of the 3H/14C ratio of intermediates with the 3H/14C ratio of T following incubations with two labeled precursors, it appeared that T is formed more readily from pregnenolone than progesterone; from 17-hydroxypregnenolone than pregnenolone, progesterone or 17-hydroxyprogesterone; and from DHA than 17-hydroxyprogesterone or 17-hydroxypregnenolone. Δ4-dione converted to T more efficiently than did Δ5-diol. The changing pattern of the intermediates with time suggested that for the Δ4 pathway the C17–20 lyase reaction is rate limiting and for the Δ5 pathway the 3β-hydroxysteroid dehydrogenase plus isomerase activity is rate limiting. On the basis of these studies, it is postulated that the predominant pathway from pregnenolone to T in human testes in vitro under the conditions used is as follows; pregnenolone to 17-hydroxypregnenolone to DHA to Δ5-diol to T.